Methods of treatment using activatable anti-EGFR antibodies

ABSTRACT

The present disclosure provides modified antibodies which contain an antibody or antibody fragment (AB) modified with a masking moiety (MM). Such modified antibodies can be further coupled to a cleavable moiety (CM), resulting in activatable antibodies (AAs), wherein the CM is capable of being cleaved, reduced, photolysed, or otherwise modified. AAs can exhibit an activatable conformation such that the AB is more accessible to a target after, for example, removal of the MM by cleavage, reduction, or photolysis of the CM in the presence of an agent capable of cleaving, reducing, or photolysing the CM. The disclosure further provides methods of making and using such modified antibodies and activatable antibodies.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/140,944, filed Apr. 28, 2016, which is a continuation of U.S. patentapplication Ser. No. 14/038,232, filed Sep. 26, 2013 and now issued asU.S. Pat. No. 9,453,078, which is a continuation of U.S. patentapplication Ser. No. 13/784,407, filed Mar. 4, 2013 and now abandoned,which is a continuation of U.S. patent application Ser. No. 13/624,293,filed Sep. 21, 2012 and now abandoned, which is a continuation of U.S.patent application Ser. No. 13/455,924, filed Apr. 25, 2012 and nowissued as U.S. Pat. No. 8,513,390, which is a continuation of U.S.patent application Ser. No. 13/315,623, filed Dec. 9, 2011 and nowissued as U.S. Pat. No. 8,563,269, which is a continuation of U.S.patent application Ser. No. 12/686,344, filed Jan. 12, 2010 and nowabandoned, which claimed the benefit of U.S. Provisional ApplicationsNos. 61/144,110, filed Jan. 12, 2009; 61/144,105, filed Jan. 12, 2009;61/249,441, filed Oct. 7, 2009; and 61/249,416, filed Oct. 7, 2009;which applications are incorporated herein by reference in theirentirety.

SEQUENCE LISTING

The contents of the text file named “CYTM-014-C08US_SeqList.txt,” whichwas created on Jul. 23, 2018 and is 197 KB, are hereby incorporated byreference in their entirety.

BACKGROUND OF THE INVENTION

Protein-based therapies have changed the face of medicine, findingapplication in a variety of different diseases. In particularantibody-based therapies have proven effective treatments for somediseases but in some cases, toxicities due to broad target expressionhave limited their therapeutic effectiveness.

As with any drugs, however, the need and desire for drugs havingimproved specificity and selectivity for their targets is of greatinterest, especially in developing second generation of antibody-baseddrugs having known targets to which they bind. Increased targeting ofantibody to the disease site could reduce systemic mechanism-basedtoxicities and lead to broader therapeutic utility.

In the realm of small molecule drugs, strategies have been developed toprovide prodrugs of an active chemical entity. Such prodrugs areadministered in a relatively inactive (or significantly less active)form. Once administered, the prodrug is metabolized in vivo into theactive compound. Such prodrug strategies can provide for increasedselectivity of the drug for its intended target and for a reduction ofadverse effects. Drugs used to target hypoxic cancer cells, through theuse of redox-activation, utilize the large quantities of reductaseenzyme present in the hypoxic cell to convert the drug into itscytotoxic form, essentially activating it. Since the prodrug has lowcytotoxicity prior to this activation, there is a markedly decreasedrisk of damage to non-cancerous cells, thereby providing for reducedside-effects associated with the drug. There is a need in the field fora strategy for providing features of a prodrug to antibody-basedtherapeutics.

SUMMARY OF THE INVENTION

The present disclosure provides for modified and activatable antibodycompositions useful for therapeutics and diagnostics. The activatableantibody compositions exhibit increased bioavailability andbiodistribution compared to conventional antibody therapeutics withprodrug features. Also provided are methods for use in diagnostics andtherapeutics, as well as screening for and construction of suchcompositions.

In one aspect, the present disclosure provides a modified antibodycomprising an antibody or antibody fragment (AB), capable ofspecifically binding its target, coupled to a masking moiety (MM),wherein the coupling of the MM reduces the ability of the AB to bind itstarget such that that the dissociation constant (K_(d)) of the ABcoupled to the MM towards the target is at least 100 times greater, atleast 1000 times greater, or at least 10,000 times greater than theK_(d) of the AB not coupled to the MM towards the target.

In another aspect, the present disclosure provides a modified antibodycomprising an antibody or antibody fragment (AB), capable ofspecifically binding its target, coupled to a masking moiety (MM),wherein the coupling of the MM to the AB reduces the ability of the ABto bind the target by at least 90%, as compared to the ability of the ABnot coupled to the MM to bind the target, when assayed in vitro using atarget displacement assay. Such coupling of the MM to the AB reduces theability of the AB to bind its target for at least 12 hours or for atleast 24 hours or for at least 72 hours.

In another aspect, the modified antibody is further coupled to acleavable moiety (CM). The CM is capable of being cleaved by an enzyme,or the CM is capable of being reduced by a reducing agent, or the CM iscapable of being photolysed. The CM is capable of being specificallycleaved, reduced, or photolysed at a rate of about at least 1×10⁴M⁻¹S⁻¹, or at least 5×10⁴ M⁻¹S, or at least 10×10⁴ M⁻¹S. In oneembodiment, the CM of the modified antibody is be within the MM.

The dissociation constant (K_(d)) of the MM towards the AB in themodified antibodies provided herein is usually at least 100 timesgreater than the K_(d) of the AB towards the target. Generally, theK_(d) of the MM towards the AB is lower than 10 nM, or lower than 5 nM,or about 1 nM.

In some embodiments, the MM of the modified antibody reduces the AB'sability to bind its target by specifically binding to theantigen-binding domain of the AB. Such binding can be non-covalent. TheMM of the modified antibody can reduce the AB's ability to bind itstarget allosterically or sterically. In specific embodiments, the MM ofthe modified antibody does not comprise more than 50% amino acidsequence similarity to a natural binding partner of the AB.

In specific embodiments, the AB of the modified antibody is an antibodyfragment that is selected from the group consisting of a Fab′ fragment,a F(ab′) 2 fragment, a scFv, a scAb, a dAb, a single domain heavy chainantibody, and a single domain light chain antibody.

In related embodiments, the AB of the modified antibody is selected fromthe group consisting of the antibodies in Table 2 or specifically thesource of the AB is cetuximab, panitumumab, infliximab, adalimumab,efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab,ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab, infliximab,bevacizumab, or figitumumab. In a specific embodiment, the modifiedantibody is not alemtuzumab.

In related embodiments, the target of the AB is selected from the groupconsisting of the targets in Table 1. In exemplary embodiments, thetarget is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20,Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1,IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4. In one specificembodiment the target is not CD52.

In a specific embodiment, the modified antibody further comprises asecond AB wherein the target for the second AB is selected from thegroup consisting of the targets in Table 1.

In related embodiments, the CM is a substrate for an enzyme selectedfrom the group consisting of the enzymes in Table 3. In specificembodiments the CM is a substrate for legumain, plasmin, TMPRSS-3/4,MMP-9, MT1-MMP, cathepsin, caspase, human neutrophil elastase,beta-secretase, uPA, or PSA. In such embodiments, where the modified ABcomprises a CM, the AB is selected from the group consisting of theantibodies in Table 2; and specifically can be from cetuximab,panitumumab, infliximab, adalimumab, efalizumab, ipilimumab,tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab,tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab,or figitumumab. In one exemplary embodiment, the AB is not alemtuzumab.

In one embodiment where the modified antibody comprises an AB, coupledto a CM and a MM, the target is selected from the group consisting ofthe targets in Table 1; or the target is EGFR, TNFalpha, CD11a, CSFR,CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4,Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44,DLL4, or IL4. In one exemplary embodiment, the target is not CD52.

The modified antibody can be further coupled to a second cleavablemoiety (CM), capable of being specifically modified by an enzyme. Inthis embodiment, the second cleavable is a substrate for legumain,plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, humanneutrophil elastase, beta-secretase, uPA, or PSA.

In another specific embodiment, the modified antibody further comprisesa linker peptide, wherein the linker peptide is positioned between theAB and the MM; or the modified antibody further comprises a linkerpeptide, wherein the linker peptide is positioned between the MM and theCM; or the modified antibody further comprises a linker peptide, whereinthe linker peptide is positioned between the AB and the CM; or themodified antibody further comprises two linker peptides, wherein thefirst linker peptide is between the AB and the CM and the second linkerpeptide is positioned between the MM and the CM. The linker is selectedfrom the group consisting of a cleavable linker, a non-cleavable linker,and a branched linker.

In certain embodiments, the modified antibody further comprises adetectable moiety. In one specific embodiment, the detectable moiety isa diagnostic agent.

In one particular embodiment, the modified antibodies described hereinfurther comprise an agent conjugated to the AB. In one aspect, the agentis a therapeutic agent, for example an antineoplastic agent. In suchembodiments, the agent is conjugated to a carbohydrate moiety of the AB,wherein the carbohydrate moiety can be located outside theantigen-binding region of the AB. Alternatively the agent is conjugatedto a sulfhydryl group of the AB.

The modified antibodies provided herein exhibit a serum half-life of atleast 5 days when administered to an organism.

The consensus sequence of the MM of some of the modified antibodiesprovided herein is CISPRGC (SEQ ID NO: 1),C(N/P)H(H/V/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H)CGCISPRGCG (SEQ ID NO: 2),xCxxYQCLxxxxxx (SEQ ID NO: 3), XXQPxPPRVXX (SEQ ID NO: 4), PxPGFPYCxxxx(SEQ ID NO: 5), xxxxQxxPWPP (SEQ ID NO: 6), GxGxCYTILExxCxxxR (SEQ IDNO: 7), GxxxCYxlxExxCxxxx (SEQ ID NO: 8), GxxxCYxlxExWCxxxx (SEQ ID NO:9), xxxCCxxYxlxxCCxxx (SEQ ID NO: 10), or xxxxxYxlLExxxxx (SEQ ID NO:11). In a specific embodiment, the consensus sequence is specific forbinding to an anti-VEGF antibody, an anti-EFGR antibody, or ananti-CTLA-4 antibody.

In a related aspect, the present disclosure provides for an activatableantibody (AA) comprising an antibody or antibody fragment (AB), capableof specifically binding its target; a masking moiety (MM) coupled to theAB, capable of inhibiting the specific binding of the AB to its target;and a cleavable moiety (CM) coupled to the AB, capable of beingspecifically cleaved by an enzyme; wherein when the AA is not in thepresence of sufficient enzyme activity to cleave the CM, the MM reducesthe specific binding of the AB to its target by at least 90% whencompared to when the AA is in the presence of sufficient enzyme activityto cleave the CM and the MM does not inhibit the specific binding of theAB to its target. In specific embodiments, the binding of the AB to itstarget is reduced for at least 12 hours, or for at least 24 hours, orfor at least 72 hours.

In one embodiment, in the AA, the dissociation constant (K_(d)) of theAB coupled to the MM and CM towards the target is at least 100 timesgreater than the K_(d) of the AB not coupled to the MM and CM towardsthe target. In a related embodiment, the dissociation constant (K_(d))of the MM towards the AB is at least 100 times greater than the K_(d) ofthe AB towards the target. Generally, the K_(d) of the MM towards the ABis lower than 10 nM, or lower than 5 nM, or about 1 nM.

In some embodiments of the AA, the MM is capable of specifically bindingto the antigen-binding domain of the AB.

In some embodiments of the AA the CM is capable of being specificallycleaved by an enzyme at a rate of about at least 1×10⁴ M⁻¹S⁻¹, or atleast 5×10⁴ M⁻¹S, or at least 10×10⁴ M⁻¹S.

In certain embodiments, of the AA where the AB is an antibody fragment,the antibody fragment is selected from the group consisting of a Fab′fragment, a F(ab′) 2 fragment, a scFv, a scAb, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody.

In certain embodiments, the AB of the AA is selected from the groupconsisting of the antibodies in Table 2. In specific embodiments, the ABis cetuximab, panitumumab, infliximab, adalimumab, efalizumab,ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab,tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab,or figitumumab.

In certain embodiments, the target of the AA is selected from the groupconsisting of the targets in Table 1. In specific embodiments, thetarget is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20,Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1,IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4.

In one specific embodiment the AB is not alemtuzumab and target is notCD52.

In certain embodiments, the CM of the AA is a substrate for legumain,plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, humanneutrophil elastase, beta-secretase, uPA, or PSA. In specificembodiments, the AA is further coupled to a second cleavable moiety(CM), capable of being specifically modified by an enzyme. In thisembodiment, the second CM is a substrate for legumain, plasmin,TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophilelastase, beta-secretase, uPA, or PSA.

In some embodiments of the AAs provided herein, the CM is located withinthe MM.

In some embodiments of the AAs provided herein, the MM does not comprisemore than 50% amino acid sequence similarity to a natural bindingpartner of the AB.

In some embodiments the AA further comprises a linker peptide, whereinthe linker peptide is positioned between the MM and the CM. In specificembodiments, the linker peptide is positioned between the AB and the CM.

In certain embodiments, the AAs provided herein further comprise adetectable moiety or an agent conjugated to the AB.

In yet another aspect, the present disclosure provides for anactivatable antibody complex (AAC) comprising: two antibodies orantibody fragments (AB1 and AB2), each capable of specifically bindingits target; at least one masking moiety (MM) coupled to either AB1 orAB2, capable of inhibiting the specific binding of AB1 and AB2 to theirtargets; and at least one cleavable moiety (CM) coupled to either AB1 orAB2, capable of being specifically cleaved by an enzyme wherebyactivating the AAC composition; wherein when the AAC is in an uncleavedstate, the MM inhibits the specific binding of AB1 and AB2 to theirtargets and when the AAC is in a cleaved state, the MM does not inhibitthe specific binding of AB1 and AB2 to their targets.

In one embodiment, the AAC is bispecific, wherein AB1 and AB2 bind thesame epitope on the same target; or the AB1 and AB2 bind to differentepitopes on the same target; or the AB1 and AB2 bind to differentepitopes on different targets.

In one embodiment of the AAC, the CM is capable of being specificallycleaved by an enzyme at a rate of about at least 1×10⁴ M⁻¹S⁻¹.

In the embodiments where AB1 or AB2 of the AAC is an antibody fragment,the antibody fragment is selected from the group consisting of a Fab′fragment, a F(ab′) 2 fragment, a scFv, a scAb, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody.

In an embodiment of the AAC, the AB1 and/or AB2 are selected from thegroup consisting of the antibodies in Table 2. In a specific embodiment,the AB1 and/or AB2 is cetuximab, panitumumab, infliximab, adalimumab,efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab,ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab, infliximab,bevacizumab, or figitumumab.

In an embodiment of the AAC, the target for the AB1 and/or AB2 isselected from the group consisting of the targets in Table 1. In arelated embodiment, the target of the AB1 and/or AB2 is EGFR, TNFalpha,CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3,Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130,VCAM-1, CD44, DLL4, or IL4. In a specific embodiment, the AB1 and AB2are capable of binding to EGFR and VEGF, a Notch Receptor and EGFR, aJagged ligand and EGFR or cMET and VEGF, respectively.

In a related AAC embodiment, the CM is a substrate for an enzymeselected from the group consisting of the enzymes in Table 3. In aspecific embodiment, the CM is a substrate for legumain, plasmin,TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophilelastase, beta-secretase, uPA, or PSA. In yet another specificembodiment, the AAC is further coupled to a second cleavable moiety(CM), capable of being specifically cleaved by an enzyme and the secondCM is a substrate for legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP,cathepsin, caspase, human neutrophil elastase, beta-secretase, uPA, orPSA.

In specific embodiments of the AAC, the MM does not comprise more than50% amino acid sequence similarity to a natural binding partner of theAB.

In other specific embodiments of the AAC, the AAC further comprises adetectable moiety or is further conjugated to an agent.

Also provided herein is a method of treating or diagnosing a conditionin a subject including administering to the subject a compositioncomprising: an antibody or antibody fragment (AB), capable ofspecifically binding its target; a masking moiety (MM) coupled to theAB, capable of inhibiting the specific binding of the AB to its target;and a cleavable moiety (CM) coupled to the AB, capable of beingspecifically cleaved by an enzyme; wherein upon administration to thesubject, when the AA is not in the presence of sufficient enzymeactivity to cleave the CM, the MM reduces the specific binding of the ABto its target by at least 90% when compared to when the AA is in thepresence of sufficient enzyme activity to cleave the CM and the MM doesnot inhibit the specific binding of the AB to its target.

In this method, the AB is selected from the group consisting of a Fab′fragment, a F(ab′) 2 fragment, a scFv, a scAb, a dAb, a single domainheavy chain antibody, and a single domain light chain antibody.

In as specific embodiment, the condition is cancer.

In another specific embodiment, the MM is not the natural bindingpartner of the AB.

In various embodiments of the method, the AB is selected from the groupconsisting of the antibodies in Table 2. Specifically in someembodiments, the AB is cetuximab, panitumumab, infliximab, adalimumab,efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8, alemtuzumab,ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab, infliximab,bevacizumab, or figitumumab.

In various embodiments of the method, the target is selected from thegroup of targets in Table 1. In specific embodiments, the target isEGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1,Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R,transferrin, gp130, VCAM-1, CD44, DLL4, or IL4.

In a very specific embodiment of the method the AB is not alemtuzumaband the target is not CD52.

In various embodiments of the method, the CM is a substrate for anenzyme selected from the group consisting of the enzymes in Table 3. Inspecific embodiments, the CM is a substrate for legumain, plasmin,TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophilelastase, beta-secretase, uPA, or PSA.

Also provided herein is a method of inhibiting angiogenesis in amammalian subject, the method comprising administering to a subject inneed thereof a therapeutically effective amount of a pharmaceuticalcomposition comprising a modified AB, an AA, an AAC, or an AACJ whereinthe target is EGFR, TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40,CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52,MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, or IL4. In aspecific embodiment, the AB is cetuximab, panitumumab, infliximab,adalimumab, efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8,alemtuzumab, ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab,infliximab, bevacizumab, or figitumumab; the CM is a substrate forlegumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, humanneutrophil elastase, beta-secretase, uPA, or PSA.

Also provided herein is a method of making an activatable antibody (AA)composition comprising: providing an antibody or antibody fragment (AB)capable of specifically binding its target; coupling a masking moiety(MM) to the AB, capable of inhibiting the specific binding of the AB toits target; and coupling a cleavable moiety (CM) to the AB, capable ofbeing specifically cleaved by an enzyme; wherein the dissociationconstant (K_(d)) of the AB coupled to the MM towards the target is atleast 100 times greater than the K_(d) of the AB not coupled to the MMtowards the target.

In one embodiment of the method, the AB is or is derived from anantibody selected from the group consisting of the antibodies in Table2. In a specific embodiment, the AB is or is derived from cetuximab,panitumumab, infliximab, adalimumab, efalizumab, ipilimumab,tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab,tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab,or figitumumab.

In one very specific embodiment, the AB is not alemtuzumab and thetarget is not CD52.

In another embodiment of the method, the CM is a substrate for an enzymeselected from the group consisting of the enzymes in Table 3. In aspecific embodiment, the CM is a substrate for legumain, plasmin,TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, human neutrophilelastase, beta-secretase, uPA, or PSA.

Also provided herein is a method of screening candidate peptides toidentify a masking moiety (MM) peptide capable of binding an antibody orantibody fragment (AB) comprising: providing a library of peptidescaffolds, wherein each peptide scaffold comprises: a transmembraneprotein (TM); and a candidate peptide; contacting an AB with thelibrary; identifying at least one candidate peptide capable of bindingthe AB; and determining whether the dissociation constant (K_(d)) of thecandidate peptide towards the AB is between 1-10 nM.

In various embodiments of the method, the library comprises viruses,cells or spores. Specifically in one embodiment, the library comprisesE. coli. In another embodiment, the peptide scaffold further comprises adetectable moiety.

Also provided is another screening method to identify a masking moiety(MM) peptide capable of masking an antibody or antibody fragment (AB)with an optimal masking efficiency comprising: providing a librarycomprising a plurality of ABs, each coupled to a candidate peptide,wherein the ABs are capable of specifically binding a target; incubatingeach library member with the target; and comparing the binding affinityof each library member towards the target with the binding affinity ofeach AB not coupled to a candidate peptide towards the target. In aspecific embodiment, the optimal binding efficiency is when the bindingaffinity of a library member to the target is 10% compared to thebinding affinity of the unmodified AB to the target.

In one aspect, also provided herein is an antibody therapeutic having animproved bioavailability wherein the affinity of binding of the antibodytherapeutic to its target is lower in a first tissue when compared tothe binding of the antibody therapeutic to its target in a secondtissue. In a related aspect, also provided herein is a pharmaceuticalcomposition comprising: an antibody or antibody fragment (AB), capableof specifically binding its target; and a pharmaceutically acceptableexcipient; wherein the affinity of the antibody or antibody fragment tothe target in a first tissue is lower than the affinity of the antibodyor antibody fragment to the target in a second tissue. In a specificembodiment, the affinity in the first tissue is 10-1,000 times lowerthan the affinity in the second tissue. In one embodiment, the AB iscoupled to a masking moiety (MM), capable of reducing the binding of theAB to its target and a cleavable moiety (CM), capable of specificallybeing cleaved by an enzyme.

In related embodiments, the target is EGFR, TNFalpha, CD11a, CSFR,CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4,Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44,DLL4, or IL4. In related embodiments, the CM is a substrate forlegumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, humanneutrophil elastase, beta-secretase, uPA, or PSA. In relatedembodiments, the antibody or antibody fragment is cetuximab,panitumumab, infliximab, adalimumab, efalizumab, ipilimumab,tremelimumab, adecatumumab, Hu5c8, alemtuzumab, ranibizumab,tositumomab, ibritumomab tiuxetan, rituximab, infliximab, bevacizumab,or figitumumab.

In a specific embodiment, the first tissue is a healthy tissue and thesecond tissue is a diseased tissue; or the first tissue is an earlystage tumor and the second tissue is a late stage tumor; the firsttissue is a benign tumor and the second tissue is a malignant tumor; orthe first tissue and second tissue are spatially separated; or the firsttissue is epithelial tissue and the second tissue is breast, head, neck,lung, pancreatic, nervous system, liver, prostate, urogenital, orcervical tissue.

In one embodiment, the antibody therapeutic is further coupled to anagent. In a specific embodiment, the agent is an antineoplastic agent.

Also provided herein are specific compositions for diagnostic andtherapeutic use. Provided herein is a composition comprising alegumain-activatable antibody or antibody fragment (AB) coupled to amasking moiety (MM); a composition comprising a plasmin-activatableantibody or antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising a caspase-activatable antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisinga TMPRSS-3/4-activatable antibody or antibody fragment (AB) coupled to amasking moiety (MM); a composition comprising a PSA-activatable antibodyor antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising a cathepsin-activatable antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisinga human neutrophil elastase-activatable antibody or antibody fragment(AB) coupled to a masking moiety (MM); a composition comprising abeta-secretase-activatable antibody or antibody fragment (AB) coupled toa masking moiety (MM); a composition comprising an uPA-activatableantibody or antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising a TMPRSS-3/4-activatable antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisinga MT1-MMP-activatable antibody or antibody fragment (AB) coupled to amasking moiety (MM); a composition comprising an activatable EGFRantibody or antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising an activatable TNFalpha antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisingan activatable CD11a antibody or antibody fragment (AB) coupled to amasking moiety (MM); a composition comprising an activatable CSFRantibody or antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising an activatable CTLA-4 antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisingan activatable EpCAM antibody or antibody fragment (AB) coupled to amasking moiety (MM); a composition comprising an activatable CD40Lantibody or antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising an activatable Notch1 antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisingan activatable Notch3 antibody or antibody fragment (AB) coupled to amasking moiety (MM); a composition comprising an activatable Jagged1antibody or antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising an activatable Jagged2 antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisingan activatable cetuximab antibody or antibody fragment (AB) coupled to amasking moiety (MM); a composition comprising an activatable vectibixantibody or antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising an activatable infliximab antibody or antibodyfragment (AB) coupled to a masking moiety (MM); a composition comprisingan activatable adalimumab antibody or antibody fragment (AB) coupled toa masking moiety (MM); a composition comprising an activatableefalizumab antibody or antibody fragment (AB) coupled to a maskingmoiety (MM); a composition comprising an activatable ipilimumab antibodyor antibody fragment (AB) coupled to a masking moiety (MM); acomposition comprising an activatable tremelimumab antibody or antibodyfragment (AB) coupled to a masking moiety (MM); or a compositioncomprising an activatable adecatumumab antibody or antibody fragment(AB) coupled to a masking moiety (MM).

INCORPORATION BY REFERENCE

All publications, patents, and patent applications mentioned in thisspecification are herein incorporated by reference to the same extent asif each individual publication, patent, or patent application wasspecifically and individually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1 shows a protease-activated AA containing an antibody (an AB), amasking moiety (MM), and a cleavable moiety (CM).

FIG. 2 shows the activity of an exemplary AA in vivo. Panel A showshealthy tissue where the AA is not able to bind, side effects areminimal; Panel B shows diseased tissue where the AA is activated by adisease-specific protease/reducing agent allowing the AA to bind totarget and be efficacious.

FIG. 3 illustrates a process to produce a protease-activated AA,involving: screening for MMs; screening for CMs; assembling the MM, CM,and an AB; expressing and purifying the assembled construct; andassaying the assembled construct for activity and toxicity in vitro andin vivo.

FIG. 4 provides an exemplary MMP-9 cleavable masked anti-VEGF scFv aminoacid sequence (SEQ ID NO: 350).

FIG. 5 provides ELISA data showing the MMP-9 activation of theMBP:anti-VEGFscFv AAs with the MMs 306 and 314. Samples were treatedwith TEV to remove the MBP fusion partner and subsequently activated byMMP-9 digestion.

FIG. 6 provides ELISA data demonstrating the MMP-9-dependent VEGFbinding of the anti-VEGFscFv His construct with the 306 MM.

FIG. 7 provides ELISA data demonstrating the MMP-9-dependent VEGFbinding of anti-VEGFscFv-Fc AAs with the MMs 306 and 314 from HEK cellsupernatants.

FIG. 8 provides ELISA data showing the MMP-9-dependent VEGF binding ofanti-VEGF scFv-Fc AA constructs with the MMs 306 and 314 that werepurified using a Protein A column.

FIG. 9 shows that the 306 MM, which binds to an anti-VEGF antibody withan affinity of >600 nM, does not efficiently preclude binding to VEGF.

FIG. 10 shows light and heavy chains of anti-CTLA4 joined via SOE-PCR togenerate scFv constructs in both orientations, V_(H)V_(L) andV_(L)V_(H). ‘(GGGS)3’ disclosed as SEQ ID NO: 102, ‘(GGGS)_(1/2)’disclosed as SEQ ID NO: 351 and ‘_(1/2)(GGGS)’ disclosed as SEQ ID NO:235.

FIG. 11 shows the use of PCR to add sites for MM cloning, CM cleavagesequence, (GGS)2 (SEQ ID NO: 111) linker on the N-terminus of theanti-CTLA4 scFv V_(H)V_(L) and V_(L)V_(H) constructs.

FIG. 12 shows the activation of an AA by MMP-9.

FIG. 13 shows that when the CM is cleaved to remove the MM, the bindingof the AB is restored.

FIG. 14 shows the activation of an AA by a protease that leads toantibody binding indistinguishable from unmodified antibodies.

FIG. 15 illustrates that an AA comprising an AB with specific bindingaffinity to VEGF is inhibited; the activated AA binds VEGF withpicomolar affinity.

FIG. 16 depicts that an AA comprising an AB with specific bindingaffinity to VEGF inhibits HUVEC proliferation.

FIG. 17 illustrates that cultured tumor cells demonstrate robust in situactivation of an AA comprising an AB with specific binding affinity toVEGF.

FIG. 18 illustrates that an AA is inactive in normal and cancer patientplasma

FIG. 19 illustrates the binding of anti-CTLA4 scFv to both murine andhuman CTLA4.

FIG. 20 shows a protease-activated AACJ-containing an antibody(containing an AB), a masking moiety (MM), a cleavable moiety (CM), anda conjugated agent. Upon cleavage of the CM and unmasking, theconjugated AB is released.

FIG. 21 shows that binding of the eCPX3.0 clones JS306, JS1825, JS1827,and JS1829 were analyzed on FACS at 3 different concentrations ofDyLight labeled anti-VEGF. All three of the affinity matured peptidesdisplayed at least 10 fold higher affinity than the JS306.

FIG. 22 shows the process for affinity maturation of some of the EGFRMM's. Consensus binding motifs disclosed as SEQ ID NOS 264-266, 264 and236, respectively, in order of appearance, and C225 binders disclosed asSEQ ID NOS 264-266, 352-355, 236, 356-360, respectively, in order ofappearance.

FIG. 23 shows the binding curves for the on-cell affinity measurement ofC225 Fab binding to MM's 3690, 3954 and 3957. MMs 3954 and 3957displayed at least 100 fold higher affinity than 3690.

FIG. 24 displays the Target Displacement Assay and extent of equilibriumbinding as a percent of parental antibody binding.

FIG. 25 shows that unlike the uPA control and substrate SM16, KK1203,1204 and 1214 show resistance to cleavage by KLK5, KLK7 and Plasmin.

FIG. 26 shows that unlike a non-optimized substrate, the optimizedsubstrates Plas1237, Plas129 and Plas 1254 show resistance to cleavageby KLK5, KLK7.

FIG. 27 Panel A shows activation of ScFv AAs containing legumainsubstrates AANL (SEQ ID NO: 361) and PTNL (SEQ ID NO: 362) followingtreatment with 5 mg/mL legumain. Panel B shows activation of ananti-VEGF IgG AA containing the legumain substrate PTNL (SEQ ID NO:362).

FIG. 28 shows the ratio of activated AA to total AA at each time pointin a legumain-activated AA. While the plasmin-activated AA is nearlycompletely activated at 7 days, both legumain-activatable AAs are onlyminimally activated. Legumain-activatable AAs isolated from serum up to7 days following injection remain masked. (n=4). ‘AANL’ disclosed as SEQID NO: 361 and ‘PTNL’ disclosed as SEQ ID NO: 362.

FIG. 29 shows that masked single-chain Fv-Fc fusion pro-antibodiesexhibit increased serum half-life.

FIG. 30 shows that the scFv-Fc serum concentration in healthy mice over10 days. The AA concentration remained stable 7 days post dose, whereasthe parent scFv-Fc concentration decreased after 3 days and was almostundetectable at 10 days.

FIG. 31 shows that AA scFv-Fc concentrations are elevated and persistlonger in serum compared with parent scFv-Fc in tumor-bearing mice. Ahigher percentage of the initial AA dose was detected in the serum at 3days (B) and 3 and 7 days (A).

FIG. 32 shows that AA scFv-Fcs persist at higher concentrations in amultidose study in Tumor-bearing mice. AAs maintained significantlyhigher serum concentrations than the parent throughout the study.

FIG. 33 shows that AAs persist at high levels in serum of normal mice ascompared to the parental antibody not modified with a MM.

FIG. 34 shows protease-activated activatable antibody complexes (AACs)containing one or more antibodies or fragments thereof (in this figurethe ABs are referred to as ABDs), a masking moiety (MM), and a cleavablemoiety (CM), where ABD1 and ABD2 are arbitrary designations for firstand second ABs. In such embodiments, the MMland MM2 bind the domainscontaining ABD land ABD2, respectively, and act as masking moieties tointerfere with target binding to an uncleaved dual target-binding AAC.The target capable of binding the ABs may be the same or differenttarget, or different binding sites of the same target. In someembodiments, binding of MM1 to the domain containing ABD2 on theopposite molecule forms the complex capable of acting as a maskingmoiety of ABD1 and ABD2.

FIG. 35 shows an AAC with cross-masking occurring such that targetbinding by both ABs is attenuated in the uncleaved state, and targetbinding is increased in the presence of an agent that cleaves the CMallowing the complex to disassemble. In this figure the AB1 and AB2 arereferred to as the ABD1 and ABD2, respectively.

FIG. 36 shows an AAC formed by covalent linkage of MM1 with ABD 1 (AB1)such that target binding by ABD2 (AB2) is attenuated in the uncleavedstate, and target binding by ABD2 (AB2) is increased in the presence ofan agent that cleaves the CM allowing the complex to disassemble. Inthis figure the AB1 and AB2 are referred to as the ABD1 and ABD2,respectively.

DETAILED DESCRIPTION OF THE INVENTION

The present disclosure provides modified antibody compositions and thatare useful for therapeutics and diagnostics. The compositions describedherein allow for greater biodistribution and improved bioavailability.

Modified and Activatable Antibodies

The modified antibody compositions described herein contain at least anantibody or antibody fragment thereof (collectively referred to as ABthroughout the disclosure), capable of specifically binding a target,wherein the AB is modified by a masking moiety (MM).

When the AB is modified with a MM and is in the presence of the target,specific binding of the AB to its target is reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target.

The K_(d) of the AB modified with a MM towards the target can be atleast 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000,100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 orgreater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000,100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 timesgreater than the K_(d) of the AB not modified with an MM or the parentalAB towards the target. Conversely, the binding affinity of the ABmodified with a MM towards the target can be at least 5, 10, 25, 50,100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000, 500,000,1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, or between5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,100,000-1,000,000, or 100,000-10,000,000 times lower than the bindingaffinity of the AB not modified with an MM or the parental AB towardsthe target.

The dissociation constant (K_(d)) of the MM towards the AB is generallygreater than the K_(d) of the AB towards the target. The K_(d) of the MMtowards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000,2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 timesgreater than the K_(d) of the AB towards the target. Conversely, thebinding affinity of the MM towards the AB is generally lower than thebinding affinity of the AB towards the target. The binding affinity ofMM towards the AB can be at least 5, 10, 25, 50, 100, 250, 500, 1,000,2,500, 5,000, 10,000, 100,000, 1,000,000 or even 10,000,000 times lowerthan the binding affinity of the AB towards the target.

When the AB is modified with a MM and is in the presence of the target,specific binding of the AB to its target can be reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target. When compared tothe binding of the AB not modified with an MM or the binding of theparental AB to the target, the AB's ability to bind the target whenmodified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4,6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96, hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12months or greater when measured in vivo or in a Target Displacement invitro immunoabsorbant assay, as described herein.

The MM can inhibit the binding of the AB to the target. The MM can bindthe antigen binding domain of the AB and inhibit binding of the AB toits target. The MM can sterically inhibit the binding of the AB to thetarget. The MM can allosterically inhibit the binding of the AB to itstarget. In these embodiments when the AB is modified or coupled to a MMand in the presence of target, there is no binding or substantially nobinding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,or 50% binding of the AB to the target, as compared to the binding ofthe AB not modified with an MM, the parental AB, or the AB not coupledto an MM to the target, for at least 2, 4, 6, 8, 12, 28, 24, 30, 36, 48,60, 72, 84, 96, hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180 days,or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or greater when measuredin vivo or in a Target Displacement in vitro immunoabsorbant assay, asdescribed herein.

When an AB is coupled to or modified by a MM, the MM can ‘mask’ orreduce, or inhibit the specific binding of the AB to its target. When anAB is coupled to or modified by a MM, such coupling or modification caneffect a structural change which reduces or inhibits the ability of theAB to specifically bind its target.

An AB coupled to or modified with an MM can be represented by thefollowing formulae (in order from an amino (N) terminal region tocarboxyl (C) terminal region:(MM)-(AB)(AB)-(MM)(MM)-L-(AB)(AB)-L-(MM)where MM is a masking moiety, the AB is an antibody or antibody fragmentthereof, and the L is a linker. In many embodiments it may be desirableto insert one or more linkers, e.g., flexible linkers, into thecomposition so as to provide for flexibility.

In certain embodiments the MM is not a natural binding partner of theAB. The MM may be a modified binding partner for the AB which containsamino acid changes that at least slightly decrease affinity and/oravidity of binding to the AB. In some embodiments the MM contains no orsubstantially no homology to the AB's natural binding partner. In otherembodiments the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to the naturalbinding partner of the AB.

The present disclosure also provides activatable antibodies (AAs) wherethe AB modified by an MM can further include one or more cleavablemoieties (CM). Such AAs exhibit activatable/switchable binding, to theAB's target. AAs generally include an antibody or antibody fragment(AB), modified by or coupled to a masking moiety (MM) and a modifiableor cleavable moiety (CM). In some embodiments, the CM contains an aminoacid sequence that serves as a substrate for a protease of interest. Inother embodiments, the CM provides a cysteine-cysteine disulfide bondthat is cleavable by reduction. In yet other embodiments the CM providesa photolytic substrate that is activatable by photolysis.

A schematic of an exemplary AA is provided in FIG. 1. As illustrated,the elements of the AA are arranged so that the CM is positioned suchthat in a cleaved (or relatively active state) and in the presence of atarget, the AB binds a target, while in an uncleaved (or relativelyinactive state) in the presence of the target, specific binding of theAB to its target is reduced or inhibited. The specific binding of the ABto its target can be reduced due to the due to the inhibition or maskingof the AB's ability to specifically bind its target by the MM.

The K_(d) of the AB modified with a MM and a CM towards the target canbe at least 5, 10, 25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000,50,000, 100,000, 500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000or greater, or between 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000,10-1,000,000, 10-10,000,000, 100-1,000, 100-10,000, 100-100,000,100-1,000,000, 100-10,000,000, 1,000-10,000, 1,000-100,000,1,000-1,000,000, 1000-10,000,000, 10,000-100,000, 10,000-1,000,000,10,000-10,000,000, 100,000-1,000,000, or 100,000-10,000,000 timesgreater than the K_(d) of the AB not modified with an MM and a CM or theparental AB towards the target. Conversely, the binding affinity of theAB modified with a MM and a CM towards the target can be at least 5, 10,25, 50, 100, 250, 500, 1,000, 2,500, 5,000, 10,000, 50,000, 100,000,500,000, 1,000,000, 5,000,000, 10,000,000, 50,000,000 or greater, orbetween 5-10, 10-100, 10-1,000, 10-10,000, 10-100,000, 10-1,000,000,10-10,000,000, 100-1,000, 100-10,000, 100-100,000, 100-1,000,000,100-10,000,000, 1,000-10,000, 1,000-100,000, 1,000-1,000,000,1000-10,000,000, 10,000-100,000, 10,000-1,000,000, 10,000-10,000,000,100,000-1,000,000, or 100,000-10,000,000 times lower than the bindingaffinity of the AB not modified with an MM and a CM or the parental ABtowards the target.

When the AB is modified with a MM and a CM and is in the presence of thetarget but not in the presence of a modifying agent (for example anenzyme, protease, reduction agent, light), specific binding of the AB toits target can be reduced or inhibited, as compared to the specificbinding of the AB not modified with an MM and a CM or the parental AB tothe target. When compared to the binding of the parental AB or thebinding of an AB not modified with an MM and a CM to its target, theAB's ability to bind the target when modified with an MM and a CM can bereduced by at least 50%, 60%, 70%, 80%, 90%, 92%, 93%, 94%, 95%, 96%,97%, 98%, 99% and even 100% for at least 2, 4, 6, 8, 12, 28, 24, 30, 36,48, 60, 72, 84, 96 hours, or 5, 10, 15, 30, 45, 60, 90, 120, 150, 180days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months or greater whenmeasured in vivo or in a Target Displacement in vitro immunoabsorbantassay, as described herein.

As used herein, the term cleaved state refers to the condition of the AAfollowing modification of the CM by a protease and/or reduction of acysteine-cysteine disulfide, bond of the CM, and/or photoactivation. Theterm uncleaved state, as used herein, refers to the condition of the AAin the absence of cleavage of the CM by a protease and/or in the absencereduction of a cysteine-cysteine disulfide bond of the CM, and/or in theabsence of light. As discussed above, the term AA is used herein torefer to an AA in both its uncleaved (native) state, as well as in itscleaved state. It will be apparent to the ordinarily skilled artisanthat in some embodiments a cleaved AA may lack an MM due to cleavage ofthe CM by protease, resulting in release of at least the MM (e.g., wherethe MM is not joined to the AA by a covalent bond (e.g., a disulfidebond between cysteine residues).

By activatable or switchable is meant that the AA exhibits a first levelof binding to a target when in a inhibited, masked or uncleaved state(i.e., a first conformation), and a second level of binding to thetarget in the uninhibited, unmasked and/or cleaved state (i.e., a secondconformation), where the second level of target binding is greater thanthe first level of binding. In general, the access of target to the ABof the AA is greater in the presence of a cleaving agent capable ofcleaving the CM than in the absence of such a cleaving agent. Thus, whenthe AA is in the uncleaved state, the AB is inhibited from targetbinding and can be masked from target binding (i.e., the firstconformation is such the AB can not bind the target), and in the cleavedstate the AB is not inhibited or is unmasked to target binding.

The CM and AB of the AA may be selected so that the AB represents abinding moiety for a target of interest, and the CM represents asubstrate for a protease that is co-localized with the target at atreatment site in a subject. Alternatively or in addition, the CM is acysteine-cysteine disulfide bond that is cleavable as a result ofreduction of this disulfide bond. AAs contain at least one of aprotease-cleavable CM or a cysteine-cysteine disulfide bond, and in someembodiments include both kinds of CMs. The AAs can alternatively orfurther include a photolabile substrate, activatable by a light source.The AAs disclosed herein find particular use where, for example, aprotease capable of cleaving a site in the CM is present at relativelyhigher levels in target-containing tissue of a treatment site (forexample diseased tissue; for example for therapeutic treatment ordiagnostic treatment) than in tissue of non-treatment sites (for examplein healthy tissue), as exemplified in FIG. 2. The AAs disclosed hereinalso find particular use where, for example, a reducing agent capable ofreducing a site in the CM is present at relatively higher levels intarget-containing tissue of a treatment or diagnostic site than intissue of non-treatment non-diagnostic sites. The AAs disclosed hereinalso find particular use where, for example, a light source, forexample, by way of laser, capable of photolysing a site in the CM isintroduced to a target-containing tissue of a treatment or diagnosticsite.

In some embodiments AAs can provide for reduced toxicity and/or adverseside effects that could otherwise result from binding of the AB atnon-treatment sites if the AB were not masked or otherwise inhibitedfrom binding its target. Where the AA contains a CM that is cleavable bya reducing agent that facilitates reduction of a disulfide bond, the ABsof such AAs may selected to exploit activation of an AB where a targetof interest is present at a desired treatment site characterized byelevated levels of a reducing agent, such that the environment is of ahigher reduction potential than, for example, an environment of anon-treatment site.

In general, an AA can be designed by selecting an AB of interest andconstructing the remainder of the AA so that, when conformationallyconstrained, the MM provides for masking of the AB or reduction ofbinding of the AB to its target. Structural design criteria to be takeninto account to provide for this functional feature.

In certain embodiments dual-target binding AAs are provided in thepresent disclosure. Such dual target binding AAs contain two ABs, whichmay bind the same or different target. In specific embodiments,dual-targeting AAs contain bispecific antibodies or antibody fragments.In one specific exemplary embodiment, the AA contains an IL17 AB and anIL23 AB. In other specific embodiments the AA contains a IL12 AB and aIL23 AB, or a EGFR AB and a VEGF AB, or a IGF1R AB and EGFR AB, or acMET AB and IGF1R AB, or a EGFR AB and a VEGF AB, or a Notch Receptor ABand a EGFR AB, or a Jagged ligand AB and a EGFR AB, or a cMET AB and aVEGF AB.

Dual target binding AAs can be designed so as to have a CM cleavable bya cleaving agent that is co-localized in a target tissue with one orboth of the targets capable of binding to the ABs of the AA. Dual targetbinding AAs with more than one AB to the same or different targets canbe designed so as to have more than one CM, wherein the first CM iscleavable by a cleaving agent in a first target tissue and wherein thesecond CM is cleavable by a cleaving agent in a second target tissue,with one or more of the targets capable of binding to the ABs of the AA.The first and second target tissues can be spatially separated, forexample, at different sites in the organism. The first and second targettissues can be the same tissue temporally separated, for example thesame tissue at two different points in time, for example the first timepoint can be when the tissue is a healthy tumor, and the second timepoint can be when the tissue is a necrosed tumor.

AAs exhibiting a switchable phenotype of a desired dynamic range fortarget binding in an inhibited versus an uninhibited conformation areprovided. Dynamic range generally refers to a ratio of (a) a maximumdetected level of a parameter under a first set of conditions to (b) aminimum detected value of that parameter under a second set ofconditions. For example, in the context of an AA, the dynamic rangerefers to the ratio of (a) a maximum detected level of target proteinbinding to an AA in the presence of protease capable of cleaving the CMof the AA to (b) a minimum detected level of target protein binding toan AA in the absence of the protease. The dynamic range of an AA can becalculated as the ratio of the equilibrium dissociation constant of anAA cleaving agent (e.g., enzyme) treatment to the equilibriumdissociation constant of the AA cleaving agent treatment. The greaterthe dynamic range of an AA, the better the switchable phenotype of theAA. AAs having relatively higher dynamic range values (e.g., greaterthan 1) exhibit more desirable switching phenotypes such that targetprotein binding by the AA occurs to a greater extent (e.g.,predominantly occurs) in the presence of a cleaving agent (e.g., enzyme)capable of cleaving the CM of the AA than in the absence of a cleavingagent.

AAs can be provided in a variety of structural configurations. Exemplaryformulae for AAs are provided below. It is specifically contemplatedthat the N- to C-terminal order of the AB, MM and CM may be reversedwithin an AA. It is also specifically contemplated that the CM and MMmay overlap in amino acid sequence, e.g., such that the CM is containedwithin the MM.

For example, AAs can be represented by the following formula (in orderfrom an amino (N) terminal region to carboxyl (C) terminal region:(MM)-(CM)-(AB)(AB)—(CM)-(MM)where MM is a masking moiety, CM is a cleavable moiety, and AB is anantibody or fragment thereof. It should be noted that although MM and CMare indicated as distinct components in the formula above, in allexemplary embodiments (including formulae) disclosed herein it iscontemplated that the amino acid sequences of the MM and the CM couldoverlap, e.g., such that the CM is completely or partially containedwithin the MM. In addition, the formulae above provide for additionalamino acid sequences that may be positioned N-terminal or C-terminal tothe AA elements.

In many embodiments it may be desirable to insert one or more linkers,e.g., flexible linkers, into the AA construct so as to provide forflexibility at one or more of the MM-CM junction, the CM-AB junction, orboth. For example, the AB, MM, and/or CM may not contain a sufficientnumber of residues (e.g., Gly, Ser, Asp, Asn, especially Gly and Ser,particularly Gly) to provide the desired flexibility. As such, theswitchable phenotype of such AA constructs may benefit from introductionof one or more amino acids to provide for a flexible linker. Inaddition, as described below, where the AA is provided as aconformationally constrained construct, a flexible linker can beoperably inserted to facilitate formation and maintenance of a cyclicstructure in the uncleaved AA.

For example, in certain embodiments an AA comprises one of the followingformulae (where the formula below represent an amino acid sequence ineither N- to C-terminal direction or C— to N-terminal direction):(MM)-L₁-(CM)-(AB)(MM)-(CM)-L₁-(AB)(MM)-L₁-(CM)-L₂-(AB)cyclo[L₁-(MM)-L₂-(CM)-L₃-(AB)]wherein MM, CM, and AB are as defined above; wherein L₁, L₂, and L₃ areeach independently and optionally present or absent, are the same ordifferent flexible linkers that include at least 1 flexible amino acid(e.g., Gly); and wherein cyclo where present, the AA is in the form of acyclic structure due to the presence of a disulfide bond between a pairof cysteines in the AA. In addition, the formulae above provide foradditional amino acid sequences that may be positioned N-terminal orC-terminal to the AA elements. It should be understood that in theformula cyclo[L₁-(MM)-L₂-(CM)-L₃-(AB)], the cysteines responsible forthe disulfide bond may be positioned in the AA to allow for one or twotails, thereby generating a lasso or omega structure when the AA is in adisulfide-bonded structure (and thus conformationally constrainedstate). The amino acid sequence of the tail(s) can provide foradditional AA features, such as binding to a target receptor tofacilitate localization of the AA, increasing serum half-life of the AA,and the like. Targeting moieties (e.g., a ligand for a receptor of acell present in a target tissue) and serum half-life extending moieties(e.g., polypeptides that bind serum proteins, such as immunoglobulin(e.g., IgG) or serum albumin (e.g., human serum albumin (HSA).

Elements of Modified and Activatable Antibodies

(a) Antibodies or Antibody Fragments (Collectively Referred to as ABs)

According to the present invention, ABs directed against any antigen orhapten may be used. ABs used in the present invention may be directedagainst any determinant, e.g., tumor, bacterial, fungal, viral,parasitic, mycoplasmal, histocompatibility, differentiation and othercell membrane antigens, pathogen surface antigens, toxins, enzymes,allergens, drugs, intracellular targets, and any biologically activemolecules. Additionally, a combination of ABs reactive to differentantigenic determinants may be used.

As used herein, the AB is a full length antibody or an antibody fragmentcontaining an antigen binding domain, which is capable of binding,especially specific binding, to a target of interest, usually a proteintarget of interest. A schematic of an AA is provided in FIG. 1. In suchembodiments, the AB can be but is not limited to variable orhypervariable regions of light and/or heavy chains of an antibody(V_(L), V_(H)), variable fragments (Fv), Fab′ fragments, F(ab′) 2fragments, Fab fragments, single chain antibodies (scAb), single chainvariable regions (scFv), complementarity determining regions (CDR),domain antibodies (dAbs), single domain heavy chain immunoglobulins ofthe BHH or BNAR type, single domain light chain immunoglobulins, orother polypeptides known in the art containing an AB capable of bindingtarget proteins or epitopes on target proteins. In further embodiments,the AB may be a chimera or hybrid combination containing more than onAB, for example a first AB and a second AB such that each AB is capableof binding to the same or different target. In some embodiments, the ABis a bispecific antibody or fragment thereof, designed to bind twodifferent antigens. In some embodiments there is a first MM and CMand/or a second MM and CM coupled to the first AB and the second AB,respectively, in the activatable form.

The origin of the AB can be a naturally occurring antibody or fragmentthereof, a non-naturally occurring antibody or fragment thereof, asynthetic antibody or fragment thereof, a hybrid antibody or fragmentthereof, or an engineered antibody or fragment thereof. The antibody canbe a humanized antibody or fragment thereof.

In certain embodiments, more than one AB is contained in the AA. In someembodiments the ABs can be derived from bispecific antibodies orfragments thereof. In other embodiments the AA can be syntheticallyengineered so as to incorporate ABs derived from two differentantibodies or fragments thereof. In such embodiments, the ABs can bedesigned to bind two different targets, two different antigens, or twodifferent epitopes on the same target. An AB containing a plurality ofABs capable of binding more than one target site are usually designed tobind to different binding sites on a target or targets of interest suchthat binding of a first AB of the AA does not substantially interferewith binding of a second AB of the AA to a target. AAs containingmultiple ABs can further include multiple AB-MM units, which mayoptionally be separated by additional CMs so that upon exposure to amodifying agent, the ABs are no longer inhibited from specificallybinding their targets, or are ‘unmasked’.

In some embodiments, use of antibody fragments as sources for the ABallow permeation of target sites at an increased rate. The Fab′fragments of IgG immunoglobulins are obtained by cleaving the antibodywith pepsin [resulting in a bivalent fragment, (Fab′) 2] or with papain[resulting in 2 univalent fragments, (2 Fab)]. Parham, 1983, J. Immunol.131: 2895-2902; Lamoyi and Nisonoff, 1983, J. Immunol. Meth. 56:235-243. The bivalent (Fab′) 2 fragment can be split by mild reductionof one or a few disulfide bonds to yield univalent Fab′ fragments. TheFab and (Fab′) 2 fragments are smaller than a whole antibody, stillcontaining an AB and, therefore can permeate the target site or tissuemore easily when used as the AB. This may offer an advantage for in vivodelivery in certain embodiments because many such fragments do not crossa placental barrier. As a result, using this embodiment of the presentinvention, an AA may be delivered at an in vivo site (such as a tumor)to a pregnant female without exposing the fetus.

Methods for generating an antibody (or fragment thereof) for a giventarget are well known in the art. The structure of antibodies andfragments thereof, variable regions of heavy and light chains of anantibody (V_(H) and V_(L)), Fv, F(ab′) 2, Fab fragments, single chainantibodies (scAb), single chain variable regions (scFv), complementaritydetermining regions (CDR), and domain antibodies (dAbs) are wellunderstood. Methods for generating a polypeptide having a desiredantigen-binding domain of a target antigen are known in the art.

Methods for modifying antibodies or antibody fragments to coupleadditional polypeptides are also well-known in the art. For instance,peptides such as MMs, CMs or linkers may be coupled to modify antibodiesto generate the modified ABs and AAs of the disclosure. AAs that containprotease-activated ABs can be developed and produced with standardmethods, as described in the schematic in FIG. 3.

The antibody or fragment thereof (collectively referred to as AB) iscapable of specifically binding a protein target. An AB of the inventioncan specifically bind to its target with a dissociation constant (K_(d))of no more than 1000 nM, 100 nM, 50 nM, 10 nM, 5 nM, 1 nM, 500 pM, 400pM, 350 pM, 300 pM, 250 pM, 200 pM, 150 pM, 100 pM, 50 pM, 25 pM, 10 pM,5 pM, 1 pM, 0.5 pM, or 0.1 pM.

Exemplary classes of targets of an AB include, but are not necessarilylimited to, cell surface receptors and secreted binding proteins (e.g.,growth factors), soluble enzymes, structural proteins (e.g. collagen,fibronectin) and the like. In some embodiments, AAs contemplated by thepresent disclosure are those having an AB capable of binding anextracellular target, usually an extracellular protein target. In otherembodiments AAs can be designed such that they are capable of cellularuptake and are designed to be switchable inside a cell.

In exemplary embodiments, in no way limiting, the AB is a bindingpartner for any target listed in Table 1. In specific exemplaryembodiments, the AB is a binding partner for EGFR, TNFalpha, CD11a,CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1,CD44, DLL4, or IL4. In one specific embodiment the AB is not a bindingpartner for CD52.

In exemplary embodiments, in no way limiting, exemplary sources for ABsare listed in Table 2. In specific exemplary embodiments, the source foran AB of the invention is cetuximab, panitumumab, infliximab,adalimumab, efalizumab, ipilimumab, tremelimumab, adecatumumab, Hu5c8,alemtuzumab, ranibizumab, tositumomab, ibritumomab tiuxetan, rituximab,infliximab, bevacizumab, or figitumumab. In one specific embodiment, thesource for the AB is not alemtuzumab or is not Campath™.

TABLE 1 Exemplary Targets 1-92-LFA-3 cMet HGF IL4 PSMA Anti-Lewis-YCollagen hGH IL4R RAAG12 Apelin J receptor CSFR Hyaluronidase IL6Sphingosine 1 Phosphate C5 complement CSFR-1 IFNalpha Insulin ReceptorTGFbeta CD11a CTLA-4 IFNbeta Jagged Ligands TNFalpha CD172A CXCR4IFNgamma Jagged 1 TNFalpha CD19 DL44 IgE Jagged 2 TNFR CD20 DLL4 IgEReceptor MUC1 TRAIL-R1 CD22 EGFR IGF Na/K ATPase TRAIL-R2 CD25 EpCAMIGF1R NGF Transferrin CD28 EPHA2 IL11 Notch Receptors Transferrinreceptor CD3 ERBB3 IL12 Notch 1 TRK-A CD30 F protein of RSV IL13 Notch 2TRK-B CD33 FAP IL15 Notch 3 VCAM-1 CD40 FGF-2 IL17 Notch 4 VEGF CD40LFGFR1 IL18 PDGF-AA VEGF-A CD41 FGFR2 IL1B PDGF-BB VEGF-B CD44 FGFR3 IL1RPDGFRalpha VEGF-C CD52 FGFR4 IL2 PDGFRalpha VEGF-D CD64 Folate receptorIL21 PDGFRbeta VEGFR1 CD80 GP IIb/IIIa IL23 PDGFRbeta VEGFR2 receptorsCD86 Gp130 IL23R Phosphatidylserine VEGFR3 CLAUDIN-3 GPIIB/IIIA IL29PlGF alpha4beta1 integrin CLAUDIN-4 HER2/neu IL2R PSCA alpha4beta7integrin

TABLE 2 Exemplary sources for ABs Antibody Trade Name (antibody name)Target Avastin ™ (bevacizumab) VEGF Lucentis ™ (ranibizumab) VEGFErbitux ™ (cetuximab) EGFR Vectibix ™ (panitumumab) EGFR Remicade ™(infliximab) TNFα Humira ™ (adalimumab) TNFα Tysabri ™ (natalizumab)Integrinα4 Simulect ™ (basiliximab) IL2R Soliris ™ (eculizumab)Complement C5 Raptiva ™ (efalizumab) CD11a Bexxar ™ (tositumomab) CD20Zevalin ™ (ibritumomab tiuxetan) CD20 Rituxan ™ (rituximab) CD20Zenapax ™ (daclizumab) CD25 Myelotarg ™ (gemtuzumab) CD33 Mylotarg ™(gemtuzumab ozogamicin) CD33 Campath ™ (alemtuzumab) CD52 ReoPro ™(abiciximab) Glycoprotein receptor IIb/IIIa Xolair ™ (omalizumab) IgEHerceptin ™ (trastuzumab) Her2 Synagis ™ (palivizumab) F protein of RSV(ipilimumab) CTLA-4 (tremelimumab) CTLA-4 Hu5c8 CD40L (pertuzumab)Her2-neu (ertumaxomab) CD3/Her2-neu Orencia ™ (abatacept) CTLA-4(tanezumab) NGF (bavituximab) Phosphatidylserine (zalutumumab) EGFR(mapatumumab) Tumor Necrosis Factor- Related Apoptosis- Inducing LigandReceptor-1 (TRAIL-R1) (matuzumab) EGFR (nimotuzumab) EGFR ICR62 EGFR mAb528 EGFR CH806 EGFR MDX-447 EGFR/CD64 (edrecolomab) EpCAM RAV12 RAAG12huJ591 PSMA Enbrel ™ (etanercept) TNF-R Amevive ™ (alefacept) 1-92-LFA-3Antril ™, Kineret ™ (ankinra) IL-1Ra GC1008 TGFbeta Notch 1 Jagged 1(adecatumumab) EpCAM (figitumumab) IGF1R (tocilizumab) IL-6

The exemplary sources for some of the ABs listed in Table 2 are detailedin the following references which are incorporated by reference hereinfor their description of one or more of the referenced AB sources:Remicade™ (infliximab): U.S. Pat. No. 6,015,557, Nagahira K, Fukuda Y,Oyama Y, Kurihara T, Nasu T, Kawashima H, Noguchi C, Oikawa S, NakanishiT. Humanization of a mouse neutralizing monoclonal antibody againsttumor necrosis factor-alpha (TNF-alpha). J Immunol Methods. 1999 Jan. 1;222(1-2):83-92.) Knight D M, Trinh H, Le J, Siegel S, Shealy D,McDonough M, Scallon B, Moore M A, Vilcek J, Daddona P, et al.Construction and initial characterization of a mouse-human chimericanti-TNF antibody. Mol Immunol. 1993 November; 30(16):1443-53. Humira™(adalimumab): Sequence in U.S. Pat. No. 6,258,562. Raptiva™(efalizumab): Sequence listed in Werther W A, Gonzalez T N, O'Connor SJ, McCabe S, Chan B, Hotaling T, Champe M, Fox J A, Jardieu P M, BermanP W, Presta L G. Humanization of an anti-lymphocyte function-associatedantigen (LFA)-1 monoclonal antibody and reengineering of the humanizedantibody for binding to rhesus LFA-1. J Immunol. 1996 Dec. 1;157(11):4986-95. Mylotarg™ (gemtuzumab ozogamicin): (Sequence listed inCO MS, Avdalovic N M, Caron P C, Avdalovic M V, Scheinberg D A, Queen C:Chimeric and humanized antibodies with specificity for the CD33 antigen.J Immunol 148:1149, 1991) (Caron P C, Schwartz M A, Co M S, Queen C,Finn R D, Graham M C, Divgi C R, Larson S M, Scheinberg D A. Murine andhumanized constructs of monoclonal antibody M195 (anti-CD33) for thetherapy of acute myelogenous leukemia. Cancer. 1994 Feb. 1; 73(3Suppl):1049-56). Soliris™ (eculizumab):_Hillmen P, Young N, Schubert J,Brodsky R, Socié G, Muus P, Roth A, Szer J, Elebute M, Nakamura R,Browne P, Risitano A, Hill A, Schrezenmeier H, Fu C, Maciejewski J,Rollins S, Mojcik C, Rother R, Luzzatto L (2006). The complementinhibitor eculizumab in paroxysmal nocturnal hemoglobinuria. N Engl JMed 355 (12): 1233-43. Tysabri™ (natalizumab): Sequence listed in LegerO J, Yednock T A, Tanner L, Homer H C, Hines D K, Keen S, Saldanha J,Jones S T, Fritz L C, Bendig M M. Humanization of a mouse antibodyagainst human alpha-4 integrin: a potential therapeutic for thetreatment of multiple sclerosis. Hum Antibodies. 1997; 8(1):3-16.Synagis™ (palivizumab): Sequence listed in Johnson S, Oliver C, Prince GA, Hemming V G, Pfarr D S, Wang S C, Dormitzer M, O'Grady J, Koenig S,Tamura J K, Woods R, Bansal G, Couchenour D, Tsao E, Hall W C, Young JF. Development of a humanized monoclonal antibody (MEDI-493) with potentin vitro and in vivo activity against respiratory syncytial virus. JInfect Dis. 1997 November; 176(5):1215-24. Ipilimumab: Immunother: 2007;30(8): 825-830 Ipilimumab (Anti-CTLA4 Antibody) Causes Regression ofMetastatic Renal Cell Cancer Associated With Enteritis and Hypophysitis;James C. Yang, Marybeth Hughes, Udai Kammula, Richard Royal, Richard M.Sherry, Suzanne L. Topalian, Kimberly B. Suri, Catherine Levy, TamikaAllen, Sharon Mavroukakis, Israel Lowy, Donald E. White, and Steven A.Rosenberg. Tremelimumab: Oncologist 2007; 12; 153-883; BlockingMonoclonal Antibody in Clinical Development for Patients with Cancer;Antoni Ribas, Douglas C. Hanson, Dennis A. Noe, Robert Millham, DeborahJ. Guyot, Steven H. Bemstein, Paul C. Canniff, Amarnath Sharma and JesusGomez-Navarro.

(b) Masking Moiety (MM)

The masking moiety (MM) of the present disclosure generally refers to anamino acid sequence coupled to the AB and positioned such that itreduces the AB's ability to specifically bind its target. In some casesthe MM is coupled to the AB by way of a linker.

When the AB is modified with a MM and is in the presence of the target,specific binding of the AB to its target is reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target.

The K_(d) of the AB modified with a MM towards the AB's target isgenerally greater than the K_(d) of the AB not modified with a MM or theK_(d) of parental AB towards the target. Conversely, the bindingaffinity of the AB modified with a MM towards the target is generallylower than the binding affinity of the AB not modified with a MM or theparental AB towards the target.

The dissociation constant (K_(d)) of the MM towards the AB is generallygreater than the K_(d) of the AB towards the target. Conversely, thebinding affinity of the MM towards the AB is generally lower than thebinding affinity of the AB towards the target.

When the AB is modified with a MM and is in the presence of the target,specific binding of the AB to its target can be reduced or inhibited, ascompared to the specific binding of the AB not modified with an MM orthe specific binding of the parental AB to the target. When the AB ismodified with a CM and a MM and is in the presence of the target but notsufficient enzyme or enzyme activity to cleave the CM, specific bindingof the modified AB to the target is reduced or inhibited, as compared tothe specific binding of the AB modified with a CM and a MM in thepresence of the target and sufficient enzyme or enzyme activity tocleave the CM.

The MM can inhibit the binding of the AB to the target. The MM can bindthe antigen binding domain of the AB and inhibit binding of the AB toits target. The MM can sterically inhibit the binding of the AB to thetarget. The MM can allosterically inhibit the binding of the AB to itstarget. In these embodiments when the AB is modified or coupled to a MMand in the presence of target, there is no binding or substantially nobinding of the AB to the target, or no more than 0.001%, 0.01%, 0.1%,1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 15%, 20%, 25%, 30%, 35%, 40%,or 50% binding of the AB to the target, as compared to the binding ofthe AB not modified with an MM, the binding of the parental AB, or thebinding of the AB not coupled to an MM to the target, for at least 2, 4,6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96 hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12months or greater when measured in vivo or in a Target Displacement invitro immunoabsorbant assay, as described herein.

In certain embodiments the MM is not a natural binding partner of theAB. The MM may be a modified binding partner for the AB which containsamino acid changes that at least slightly decrease affinity and/oravidity of binding to the AB. In some embodiments the MM contains no orsubstantially no homology to the AB's natural binding partner. In otherembodiments the MM is no more than 5%, 10%, 15%, 20%, 25%, 30%, 35%,40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, or 80% similar to the naturalbinding partner of the AB.

When the AB is in a ‘masked’ state, even in the presence of a target forthe AB, the MM interferes with or inhibits the binding of the AB to thetarget. However, in the unmasked state of the AB, the MM's interferencewith target binding to the AB is reduced, thereby allowing greateraccess of the AB to the target and providing for target binding.

For example, when the modified antibody is an AA and comprises a CM, theAB can be unmasked upon cleavage of the CM, in the presence of enzyme,preferably a disease-specific enzyme. Thus, the MM is one that when theAA is uncleaved provides for masking of the AB from target binding, butdoes not substantially or significantly interfere or compete for bindingof the target to the AB when the AA is in the cleaved conformation.Thus, the combination of the MM and the CM facilitates theswitchable/activatable phenotype, with the MM decreasing binding oftarget when the AA is uncleaved, and cleavage of the CM by proteaseproviding for increased binding of target.

The structural properties of the MM will vary according to a variety offactors such as the minimum amino acid sequence required forinterference with AB binding to target, the target protein-AB bindingpair of interest, the size of the AB, the length of the CM, whether theCM is positioned within the MM and also serves to mask the AB in theuncleaved AA, the presence or absence of linkers, the presence orabsence of a cysteine within or flanking the AB that is suitable forproviding a CM of a cysteine-cysteine disulfide bond, and the like.

One strategy for masking an antibody or fragment thereof (AB) in an AAis to provide the AA in a loop that sterically hinders access of targetto the AB. In this strategy, cysteines are positioned at or near theN-terminus, C-terminus, or AB of the AA, such that upon formation of adisulfide bond between the cysteines, the AB is masked.

In some embodiments, the MM is coupled to the AA by covalent binding. Inanother embodiment, the AA composition is prevented from binding to thetarget by binding the MM to an N-terminus of the AA. In yet anotherembodiment, the AA is coupled to the MM by cysteine-cysteine disulfidebridges between the MM and the AA.

The MM can be provided in a variety of different forms. In certainembodiments, the MM can be selected to be a known binding partner of theAB, provided that the MM binds the AB with less affinity and/or aviditythan the target protein to which the AB is designed to bind followingcleavage of the CM so as to reduce interference of MM in target-ABbinding. Stated differently, as discussed above, the MM is one thatmasks the AB from target binding when the AA is uncleaved, but does notsubstantially or significantly interfere or compete for binding fortarget when the AA is in the cleaved conformation. In a specificembodiment, the AB and MM do not contain the amino acid sequences of anaturally-occurring binding partner pair, such that at least one of theAB and MM does not have the amino acid sequence of a member of anaturally occurring binding partner

The efficiency of the MM to inhibit the binding of the AB to its targetwhen coupled can be measured by a Masking Efficiency measure, using animmunoabsorbant Target Displacement Assay, as described herein in theExamples section of the disclosure. Masking efficiency of MMs isdetermined by at least two parameters: affinity of the MM for theantibody or fragment thereof and the spatial relationship of the MMrelative to the binding interface of the AB to its target.

Regarding affinity, by way of example, an MM may have high affinity butonly partially inhibit the binding site on the AB, while another MM mayhave a lower affinity for the AB but fully inhibit target binding. Forshort time periods, the lower affinity MM may show sufficient masking;in contrast, over time, that same MM may be displaced by the target (dueto insufficient affinity for the AB).

In a similar fashion, two MMs with the same affinity may show differentextents of masking based on how well they promote inhibition of thebinding site on the AB or prevention of the AB from binding its target.In another example, a MM with high affinity may bind and change thestructure of the AB so that binding to its target is completelyinhibited while another MM with high affinity may only partially inhibitbinding. As a consequence, discovery of an effective MM cannot be basedonly on affinity but can include an empirical measure of MaskingEfficiency. The time-dependent target displacement of the MM in the AAcan be measured to optimize and select for MMs. A novel TargetDisplacement Assay is described herein for this purpose.

In some embodiments the MM can be identified through a screeningprocedure from a library of candidates AAs having variable MMs. Forexample, an AB and CM can be selected to provide for a desiredenzyme/target combination, and the amino acid sequence of the MM can beidentified by the screening procedure described below to identify an MMthat provides for a switchable phenotype. For example, a random peptidelibrary (e.g., from about 2 to about 40 amino acids or more) may be usedin the screening methods disclosed herein to identify a suitable MM. Inspecific embodiments, MMs with specific binding affinity for an antibodyor fragment thereof (AB) can be identified through a screening procedurethat includes providing a library of peptide scaffolds consisting ofcandidate MMs wherein each scaffold is made up of a transmembraneprotein and the candidate MM. The library is then contacted with anentire or portion of an AB such as a full length antibody, a naturallyoccurring antibody fragment, or a non-naturally occurring fragmentcontaining an AB (also capable of binding the target of interest), andidentifying one or more candidate MMs having detectably bound AB.Screening can include one more rounds of magnetic-activated sorting(MACS) or fluorescence-activated sorting (FACS). Screening can alsoincluded determination of the dissociation constant (K_(d)) of MMtowards the AB and subsequent determination of the Masking Efficiency.

In this manner, AAs having an MM that inhibits binding of the AB to thetarget in an uncleaved state and allows binding of the AB to the targetin a cleaved state can be identified, and can further provide forselection of an AA having an optimal dynamic range for the switchablephenotype. Methods for identifying AAs having a desirable switchingphenotype are described in more detail below.

Alternatively, the MM may not specifically bind the AB, but ratherinterfere with AB-target binding through non-specific interactions suchas steric hindrance. For example, the MM may be positioned in theuncleaved AA such that the tertiary or quaternary structure of the AAallows the MM to mask the AB through charge-based interaction, therebyholding the MM in place to interfere with target access to the AB.

AAs can also be provided in a conformationally constrained structure,such as a cyclic structure, to facilitate the switchable phenotype. Thiscan be accomplished by including a pair of cysteines in the AA constructso that formation of a disulfide bond between the cysteine pairs placesthe AA in a loop or cyclic structure. Thus the AA remains cleavable bythe desired protease while providing for inhibition of target binding tothe AB. Upon cleavage of the CM, the cyclic structure is opened,allowing access of target to the AB.

The cysteine pairs can be positioned in the AA at any position thatprovides for a conformationally constrained AA, but that, following CMreduction, does not substantially or significantly interfere with targetbinding to the AB. For example, the cysteine residues of the cysteinepair are positioned in the MM and a linker flanked by the MM and AB,within a linker flanked by the MM and AB, or other suitableconfigurations. For example, the MM or a linker flanking an MM caninclude one or more cysteine residues, which cysteine residue forms adisulfide bridge with a cysteine residue positioned opposite the MM whenthe AA is in a folded state. It is generally desirable that the cysteineresidues of the cysteine pair be positioned outside the AB so as toavoid interference with target binding following cleavage of the AA.Where a cysteine of the cysteine pair to be disulfide bonded ispositioned within the AB, it is desirable that it be positioned to as toavoid interference with AB-target binding following exposure to areducing agent.

Exemplary AAs capable of forming a cyclic structure by disulfide bondsbetween cysteines can be of the general formula (which may be fromeither N- to C-terminal or from C— to N-terminal direction):X_(n1)-(Cys₁)—X_(m)-CM-AB—(Cys₂)—X_(n2)X_(n1)-cyclo[(Cys₁)—X_(m)-CM-AB—(Cys₂)]—X_(n2)wherein

X_(n1) and X_(n2) are independently, optionally present or absent and,when present, independently represent any amino acid, and can optionallyinclude an amino acid sequence of a flexible linker (e.g., at least oneGly, Ser, Asn, Asp, usually at least one Gly or Ser, usually at leastone Gly), and n₁ and n₂ are independently selected from s zero or anyinteger, usually nor more than 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10;

Cys₁ and Cys₂ represent first and second cysteines of a pair capable offorming a disulfide bond;

X_(m) represents amino acids of a masking motif (MM), where X is anyamino acid, wherein X_(m) can optionally include a flexible linker(e.g., at least one Gly, Ser, Asn, Asp, usually at least one Gly or Ser,usually at least one Gly); and where m is an integer greater than 1,usually 2, 3, 4, 5, 6, 7, 8, 9, 10 or more (as described above);

CM represents a cleavable moiety (as described herein); and

AB represents an antibody or fragment thereof (as described herein).

As used in the formula above, cyclo indicates a disulfide bond in the AAthat provides for a cyclic structure of the AA. Furthermore, the formulaabove contemplate dual target-binding AAs wherein MM refers to an AB1and AB refers to AB2, where AB1 and AB2 are arbitrary designations forfirst and second ABs, and where the target capable of binding the ABsmay be the same or different target, or the same or different bindingsites of the same target. In such embodiments, the AB1 and/or AB2 actsas a masking moiety to interfere with target binding to an uncleaveddual target-binding AA.

As illustrated above, the cysteines can thus be positioned in the AAallow for one or two tails (represented by X_(n1) and X_(n2) above),thereby generating a lasso or omega structure when the AA is in adisulfide-bonded structure (and thus conformationally constrainedstate). The amino acid sequence of the tail(s) can provide foradditional AA features, such as binding to a target receptor tofacilitate localization of the AA.

In certain specific embodiments, the MM does not inhibit cellular entryof the AA.

(c) Cleavable Moiety (CM)

In some embodiments, the cleavable moiety (CM) of the AA may include anamino acid sequence that can serve as a substrate for a protease,usually an extracellular protease. In other embodiments, the CMcomprises a cysteine-cysteine pair capable of forming a disulfide bond,which can be cleaved by action of a reducing agent. In other embodimentsthe CM comprises a substrate capable of being cleaved upon photolysis.

The CM is positioned in the AA such that when the CM is cleaved by acleaving agent (e.g., a protease substrate of a CM is cleaved by theprotease and/or the cysteine-cysteine disulfide bond is disrupted viareduction by exposure to a reducing agent) or by light-inducedphotolysis, in the presence of a target, resulting in a cleaved state,the AB binds the target, and in an uncleaved state, in the presence ofthe target, binding of the AB to the target is inhibited by the MM (FIG.2). It should be noted that the amino acid sequence of the CM mayoverlap with or be included within the MM, such that all or a portion ofthe CM facilitates masking of the AB when the AA is in the uninhibitedor uncleaved or unmasked conformation.

The CM may be selected based on a protease that is co-localized intissue with the desired target of the AB of the AA. A variety ofdifferent conditions are known in which a target of interest isco-localized with a protease, where the substrate of the protease isknown in the art. In the example of cancer, the target tissue can be acancerous tissue, particularly cancerous tissue of a solid tumor. Thereare reports in the literature of increased levels of proteases havingknown substrates in a number of cancers, e.g., solid tumors. See, e.g.,La Rocca et al, (2004) British J. of Cancer 90(7): 1414-1421. Non-limingexamples of disease include: all types of cancers (breast, lung,colorectal, prostate, head and neck, pancreatic, etc), rheumatoidarthritis, Crohn's disase, melanomas, SLE, cardiovascular damage,ischemia, etc. Furthermore, anti-angiogenic targets, such as VEGF, areknown. As such, where the AB of an AA is selected such that it iscapable of binding an anti-angiogenic target such as VEGF, a suitable CMwill be one which comprises a peptide substrate that is cleavable by aprotease that is present at the cancerous treatment site, particularlythat is present at elevated levels at the cancerous treatment site ascompared to non-cancerous tissues. In one exemplary embodiment, the ABof an AA can bind VEGF and the CM can be a matrix metalloprotease (MMP)substrate, and thus is cleavable by an MMP. In other embodiments, the ABof an AA can bind a target of interest and the CM can be, for example,legumain, plasmin, TMPRSS-3/4, MMP-9, MT1-MMP, cathepsin, caspase, humanneutrophil elastase, beta-secretase, uPA, or PSA. In other embodiments,the AA is activated by other disease-specific proteases, in diseasesother than cancer such as multiple sclerosis or rheumatoid arthritis.

The unmodified or uncleaved CM can allow for efficient inhibition ormasking of the AB by tethering the MM to the AB. When the CM is modified(cleaved, reduced, photolysed), the AB is no longer inhibited orunmasked and can bind its target.

The AA can comprise more than one CM such that the AA would comprise,for example, a first CM (CM1) and a second CM (CM2). The CM1 and CM2 canbe different substrates for the same enzyme (for example exhibitingdifferent binding affinities to the enzyme), or different substrates fordifferent enzymes, or CM1 can be an enzyme substrate and CM2 can be aphotolysis substrate, or CM1 can be an enzyme substrate and CM2 can be asubstrate for reduction, or CM1 can be a substrate for photolysis andCM2 can be a substrate for reduction, and the like.

The CM is capable of being specifically modified (cleaved, reduced orphotolysed) by an agent (ie enzyme, reducing agent, light) at a rate ofabout 0.001-1500×10⁴ M⁻¹S⁻¹ or at least 0.001, 0.005, 0.01, 0.05, 0.1,0.5, 1, 2.5, 5, 7.5, 10, 15, 20, 25, 50, 75, 100, 125, 150, 200, 250,500, 750, 1000, 1250, or 1500×10⁴ M⁻¹S⁻¹.

For specific cleavage by an enzyme, contact between the enzyme and CM ismade. When the AA comprising an AB coupled to a MM and a CM is in thepresence of target and sufficient enzyme activity, the CM can becleaved. Sufficient enzyme activity can refer to the ability of theenzyme to make contact with the CM and effect cleavage. It can readilybe envisioned that an enzyme may be in the vicinity of the CM but unableto cleave because of other cellular factors or protein modification ofthe enzyme.

Exemplary substrates can include but are not limited to substratescleavable by one or more of the following enzymes or proteases in Table3.

TABLE 3 Exemplary Enzymes/Proteases ADAM10 Caspase 8 Cathepsin S MMP 8ADAM12 Caspase 9 FAP MMP 9 ADAM17 Caspase 10 Granzyme B MMP-13 ADAMTSCaspase 11 Guanidinobenzoatase (GB) MMP 14 ADAMTS5 Caspase 12 HepsinMT-SP1 BACE Caspase 13 Human Neutrophil Neprilysin Elastase (HNE)Caspases Caspase 14 Legumain NS3/4A Caspase 1 Cathepsins Matriptase 2Plasmin Caspase 2 Cathepsin A Meprin PSA Caspase 3 Cathepsin B MMP 1PSMA Caspase 4 Cathepsin D MMP 2 TACE Caspase 5 Cathepsin E MMP 3 TMPRSS3/4 Caspase 6 Cathepsin K MMP 7 uPA Caspase 7 MT1-MMP

Alternatively or in addition, the AB of an AA can be one that binds atarget of interest and the CM can involve a disulfide bond of a cysteinepair, which is thus cleavable by a reducing agent such as, for example,but not limited to a cellular reducing agent such as glutathione (GSH),thioredoxins, NADPH, flavins, ascorbate, and the like, which can bepresent in large amounts in tissue of or surrounding a solid tumor.

(d) Linkers

Linkers suitable for use in compositions described herein are generallyones that provide flexibility of the modified AB or the AA to facilitatethe inhibition of the binding of the AB to the target. Such linkers aregenerally referred to as flexible linkers. Suitable linkers can bereadily selected and can be of any of a suitable of different lengths,such as from 1 amino acid (e.g., Gly) to 20 amino acids, from 2 aminoacids to 15 amino acids, from 3 amino acids to 12 amino acids, including4 amino acids to 10 amino acids, 5 amino acids to 9 amino acids, 6 aminoacids to 8 amino acids, or 7 amino acids to 8 amino acids, and may be 1,2, 3, 4, 5, 6, or 7 amino acids.

Exemplary flexible linkers include glycine polymers (G)_(n),glycine-serine polymers (including, for example, (GS)_(n), (GSGGS)_(n)(SEQ ID NO: 12) and (GGGS)_(n) (SEQ ID NO: 13), where n is an integer ofat least one), glycine-alanine polymers, alanine-serine polymers, andother flexible linkers known in the art. Glycine and glycine-serinepolymers are relatively unstructured, and therefore may be able to serveas a neutral tether between components. Glycine accesses significantlymore phi-psi space than even alanine, and is much less restricted thanresidues with longer side chains (see Scheraga, Rev. Computational Chem.11173-142 (1992)). Exemplary flexible linkers include, but are notlimited to Gly-Gly-Ser-Gly (SEQ ID NO: 14), Gly-Gly-Ser-Gly-Gly (SEQ IDNO: 15), Gly-Ser-Gly-Ser-Gly (SEQ ID NO: 16), Gly-Ser-Gly-Gly-Gly (SEQID NO: 17), Gly-Gly-Gly-Ser-Gly (SEQ ID NO: 18), Gly-Ser-Ser-Ser-Gly(SEQ ID NO: 19), and the like. The ordinarily skilled artisan willrecognize that design of an AA can include linkers that are all orpartially flexible, such that the linker can include a flexible linkeras well as one or more portions that confer less flexible structure toprovide for a desired AA structure.

(e) Additional Elements

In addition to the elements described above, the modified ABs and AAscan contain additional elements such as, for example, amino acidsequence N- or C-terminal of the AA. For example, AAs can include atargeting moiety to facilitate delivery to a cell or tissue of interest.Moreover, in the context of the AA libraries discussed further below,the AA can be provided in the context of a scaffold protein tofacilitate display of the AA on a cell surface.

Exemplary Embodiments

The compositions and AAs provided here in can be useful for a variety ofpurposes including therapeutics and diagnostics.

An exemplary AA provided herein can be a legumain-activatable anti-EGFRcoupled to a MM, plasmin-activatable anti-EGFR coupled to a MM,TMPRSS-3/4 activatable anti-EGFR coupled to a MM, legumain-activatablecetuximab coupled to a MM, plasmin-activatable cetuximab coupled to aMM, TMPRSS-3/4 activatable cetuximab coupled to a MM,legumain-activatable vectibix coupled to a MM, plasmin-activatablevectibix coupled to a MM, or a TMPRSS-3/4 activatable vectibix coupledto a MM. In some embodiments these AAs can be useful for the treatmentof diagnosis of head and neck carcinomas, or colon, lung, or pancreaticcarcinomas.

An exemplary AA provided herein can be a MMP9-activatable anti-TNFalphacoupled to a MM, MT1-MMP-activatable anti-TNFalpha coupled to a MM,cathepsin-activatable anti-TNFalpha coupled to a MM, MMP9-activatableinfliximab coupled to a MM, MT1-MMP-activatable infliximab coupled to aMM, cathepsin-activatable infliximab coupled to a MM, MMP9-activatableadalimumab coupled to a MM, MT1-MMP-activatable adalimumab coupled to aMM, or a cathepsin-activatable adalimumab coupled to a MM. In someembodiments these AAs can be useful for the treatment of diagnosis ofrheumatoid arthritis or multiple sclerosis.

An exemplary AA provided herein can be a legumain-activatable anti-CD11acoupled to a MM, plasmin-activatable anti-CD11a coupled to a MM,caspase-activatable anti-CD11a coupled to a MM, cathepsin-activatableanti-CD11a coupled to a MM, legumain-activatable efalizumab coupled to aMM, plasmin-activatable efalizumab coupled to a MM, caspase-activatableefalizumab coupled to a MM, cathepsin-activatable efalizumab coupled toa MM, legumain-activatable anti-CSFR coupled to a MM,plasmin-activatable anti-CSFR coupled to a MM, caspase-activatableanti-CSFR coupled to a MM, or a cathepsin-activatable anti-CSFR coupledto a MM. In some embodiments these AAs can be useful for the treatmentor diagnosis of tumor associated macrophages for carcinomas.

An exemplary AA provided herein can be a plasmin-activatable anti-CTLA-4coupled to a MM, caspase-activatable anti-CTLA-4 coupled to a MM,MT1-MMP-activatable anti-CTLA-4 coupled to a MM, plasmin-activatableipilimumab coupled to a MM, caspase-activatable ipilimumab coupled to aMM,

MT1-MMP-activatable ipilimumab coupled to a MM, plasmin-activatabletremelimumab coupled to a MM, caspase-activatable tremelimumab coupledto a MM, or a MT1-MMP-activatable tremelimumab coupled to a MM. In someembodiments these AAs can be useful for the treatment or diagnosis ofmalignant melanomas.

An exemplary AA provided herein can be a PSA-activatable anti-EPCAMcoupled to a MM, legumain-activatable anti-EPCAM coupled to a MM,PSA-activatable adecatumumab coupled to a MM or a legumain-activatableadecatumumab coupled to a MM. In some embodiments these AAs can beuseful for the treatment or diagnosis of prostate cancer.

An exemplary AA provided herein can be a human neutrophilelastase-activatable anti-CD40L coupled to a MM, or a human neutrophilelastase-activatable Hu5c8 coupled to a MM. In some embodiments theseAAs can be useful for the treatment or diagnosis of lymphomas.

An exemplary AA provided herein can be a beta-secretase-activatableanti-Notch1 coupled to a MM, legumain-activatable anti-Notch1 coupled toa MM, plasmin-activatable anti-Notch1 coupled to a MM, uPA-activatableanti-Notch1 coupled to a MM, beta-secretase-activatable anti-Notch3coupled to a MM, legumain-activatable anti-Notch3 coupled to a MM,plasmin-activatable anti-Notch3 coupled to a MM, uPA-activatableanti-Notch3 coupled to a MM, beta-secretase-activatable anti-Jagged1coupled to a MM, legumain-activatable anti-Jagged1 coupled to a MM,plasmin-activatable anti-Jagged1 coupled to a MM, uPA-activatableanti-Jagged1 coupled to a MM, beta-secretase-activatable anti-Jagged2coupled to a MM, legumain-activatable anti-Jagged2 coupled to a MM,plasmin-activatable anti-Jagged2 coupled to a MM, or a uPA-activatableanti-Jagged2 coupled to a MM. In some embodiments these AAs can beuseful for the treatment or diagnosis of triple negative (ER, PR andHer2 negative) breast, head and neck, colon and other carcinomas.

An exemplary AA provided herein can be a MMP-activatable anti-CD52coupled to a MM, or a MMP-activatable anti-campath coupled to a MM. Insome embodiments these AAs can be useful for the treatment or diagnosisof multiple sclerosis.

An exemplary AA provided herein can be a MMP-activatable anti-MUC1coupled to a MM, legumain-activatable anti-MUC 1 coupled to a MM,plasmin-activatable anti-MUC 1 coupled to a MM, or a uPA-activatableanti-MUC 1 coupled to a MM. In some embodiments these AAs can be usefulfor the treatment or diagnosis of epithelial derived tumors.

An exemplary AA provided herein can be a legumain-activatable anti-IGF1Rcoupled to a MM, plasmin-activatable anti-IGF1R coupled to a MM,caspase-activatable anti-IGF1R coupled to a MM, legumain-activatableanti-figitumumab coupled to a MM, plasmin-activatable anti-figitumumabcoupled to a MM, or a caspase-activatable anti-figitumumab coupled to aMM. In some embodiments these AAs can be useful for the treatment ordiagnosis of non-small cell lung, and other epithelial tumors.

An exemplary AA provided herein can be a legumain-activatableanti-transferrin receptor coupled to a MM, plasmin-activatableanti-transferrin receptor coupled to a MM, or a caspase-activatableanti-transferrin receptor coupled to a MM. In some embodiments these AAscan be useful for the treatment or diagnosis of solid tumors, pancreatictumors.

An exemplary AA provided herein can be a legumain-activatable anti-gp130coupled to a MM, plasmin-activatable anti-gp130 coupled to a MM, or auPA-activatable anti-gp130 coupled to a MM. In some embodiments theseAAs can be useful for the treatment or diagnosis of solid tumors.

In certain other non-limiting exemplary embodiments, activatableantibody compositions include an legumain masked AB specific for Notch1,a uPA activatable masked AB specific for Jagged1, a plasmin activatable,masked anti-VEGF scFv, a MMP-9 activatable, masked anti-VCAM scFv, and aMMP-9 activatable masked anti-CTLA4.

These AAs are provided by way of example only and such enzymeactivatable masked antibody AAs could be designed to any target aslisted in but not limited to those in Table 1 and by using any antibodyas listed in but not limited to those in Table 2.

Activatable Antibody Complexes

In one aspect of the invention, the AA exists as a complex (AAC)comprising two or more ABs, as depicted in FIGS. 34-36. The presentdisclosure provides complexes of activatable antibodies (AACs), whichexhibit activatable/switchable binding to one or more target proteins.AACs generally include one or more antibodies or antibody fragments(ABs), masking moieties (MMs), and cleavable moieties (CMs). In someembodiments, the CM contains an amino acid sequence that serves as asubstrate for a protease of interest. In other embodiments, the CMprovides a cysteine-cysteine disulfide bond that is cleavable byreduction. The AAC exhibits an activatable conformation such that atleast one AB is less accessible to target when unmodified than aftermodification of the CM, e.g., in the presence of a cleavage agent (e.g.,a protease that recognizes the cleavage site of the CM) or a reducingagent (e.g. a reducing agent that reduces disulfide bonds in the CM).

The CM and AB of the AAC may be selected so that the AB represents abinding moiety for a target of interest, and the CM represents asubstrate for a protease that is co-localized with the target at atreatment site in a subject. In some embodiments AACs can provide forreduced toxicity and/or adverse side effects that could otherwise resultfrom binding of the ABs at non-treatment sites if they were not masked.In some embodiments, the AAC can further comprise a detectable moiety ora diagnostic agent. In certain embodiments the AAC is conjugated to atherapeutic agent located outside the antigen binding region. AACs canalso be used in diagnostic and/or imaging methods or to detect thepresence or absence of a cleaving agent in a sample.

A schematic of an AAC is provided in FIG. 34. As illustrated, theelements of the AAC are arranged so that the CM is positioned such thatin a cleaved state (or relatively active state) and in the presence of atarget, the AB binds a target, while in an uncleaved state (orrelatively inactive state) in the presence of the target, binding of theABs to the target is inhibited due to the masking of the ABs by the MMin the complex. As used herein, the term cleaved state refers to thecondition of the AAC following cleavage of the CM by a protease and/orreduction of a cysteine-cysteine disulfide bond of the CM. The termuncleaved state, as used herein, refers to the condition of the AAC inthe absence of cleavage of the CM by a protease and/or in the absencereduction of a cysteine-cysteine disulfide bond of the CM. As discussedabove, the term AAC is used herein to refer to AAC in both its uncleaved(native) state, as well as in its cleaved state. It will be apparent tothe ordinarily skilled artisan that in some embodiments a cleaved AACmay lack an MM due to cleavage of the CM by protease, resulting inrelease of at least the MM (e.g., where the MM is not joined to the AACby a covalent bond.)

By activatable or switchable is meant that the AAC exhibits a firstlevel of binding to a target when in a native or uncleaved state (i.e.,a first conformation), and a second level of binding to the target inthe cleaved state (i.e., a second conformation), where the second levelof target binding is greater than the first level of binding. Ingeneral, access of target to the AB of the AAC is greater in thepresence of a cleaving agent capable of cleaving the CM than in theabsence of such a cleaving agent. Thus, in the native or uncleaved statethe AB is masked from target binding (i.e., the first conformation issuch that it interferes with access of the target to the AB), and in thecleaved state the AB is unmasked to target binding.

In general, an AAC can be designed by selecting an AB(s) of interest andconstructing the remainder of the AAC so that, when conformationallyconstrained, the MM provides for masking of the AB. Dual target bindingAACs contain two ABs, which may bind the same or different target. Inspecific embodiments, dual-targeting AACs contain bispecific antibodiesor antibody fragments.

In certain embodiments, a complex is comprised of two activatableantibodies (AA), each containing an AB, CM, and MM such thatcross-masking occurs—that is, the MM on one AA interferes with targetbinding by the AB on the other AA (FIG. 34A). In other embodiments, acomplex is comprised of two AAs, with each AA containing an AB and onecontaining a CM and MM such that universal cross-masking occurs—that is,the MM effects formation of the complex and interferes with targetbinding by the ABs on both AAs (FIG. 34B). In other embodiments, acomplex is comprised of two AAs, each containing two ABs, CMs, and MMssuch that cross-masking occurs—that is, the MMs on one AA interfere withtarget binding by the ABs on the other AA (FIG. 34C). In otherembodiments, a complex is comprised of two AAs, with one AA containingtwo ABs, CMs, and MMs such that universal cross-masking occurs—that is,the MMs interfere with target binding by the ABs on both AAs (FIG. 34D).In other embodiments, a complex is comprised of two molecules of abispecific AA where the bispecific AA contains two ABs, CMs, and MMssuch that cross-masking occurs in the complex—that is, the MM1interferes with target binding by the AB1 on the opposite molecule, andthe MM2 interferes with target binding by the AB2 on the oppositemolecule (FIG. 34E). In other embodiments, a complex is comprised of twomolecules of a bispecific AA where the bispecific AA contains two ABs,one CM, and one MM such that universal cross-masking occurs in thecomplex—that is, the MM interferes with target binding by both ABs (FIG.34F).

In general, disassembly of the AAC and access of targets to at least oneof the ABs of the AACs are greater in the presence of a cleaving agentcapable of cleaving the CMs than in the absence of such a cleaving agent(FIG. 35). The two AAs of a complex may contain ABs that bind differenttargets, or that bind different epitopes on the same target.

One of the MM/AB pairs of the complex may be used for stable complexformation and have no therapeutic target on its own. A high affinity MMfor the non-therapeutic AB allows a stable complex to form, even with alower affinity MM for the therapeutic AB. The low affinity MM for thetherapeutic AB, in the context of the multivalent complex, will besufficient for masking the therapeutic AB, but after cleavage will morereadily dissociate. For maximum target binding in the cleaved state, thedifference in affinity of the MM and target for the AB should bemaximized.

In other embodiments, an AB may form a covalent linkage to an MM on theopposite molecule of the complex. In the presence of a cleaving agentthe complex disassembles such that at least one of the other ABs willbind its target (FIG. 36). Such a covalent linkage may form betweenreactive amino acid side chains in the MM and AB, eg. disulfide bondbetween cysteines, or by chemical conjugation of reactive groups to theMM and a catalytic AB. For examples of covalent binding antibodies seeChmura A. J. et al., Proc Natl Acad Sci USA. 2001 Jul. 17, 98(15):8480-8484; Rader, C. et al., Proc Natl Acad Sci USA. 2003 Apr. 29,100(9): 5396-5400; Armentano, F. et al., Immunology Letters 2006 Feb.28, 103(1): 51-57.

It should be noted that although MM and CM are indicated as distinctcomponents, it is contemplated that the amino acid sequences of the MMand the CM could overlap, e.g., such that the CM is completely orpartially contained within the MM. In many embodiments it may bedesirable to insert one or more linkers, e.g., flexible linkers, intothe AAC construct so as to provide for flexibility at one or more of theMM-CM junction, the CM-AB junction, or both. In addition to the elementsdescribed above, the AACs can contain additional elements such as, forexample, amino acid sequence N- or C-terminal of the AAC.

Activatable Antibody Conjugates

In one aspect of the invention, the AB of the AA is further conjugatedto an agent such as a therapeutic agent, thus producing activatableantibody conjugates (AACJs), a specific type of AA. The agent isattached either directly or via a linker to the AB. Such agents orlinkers are selectively attached to those areas of ABs which are not apart of nor directly involved with the antigen binding site of themolecule. An exemplary AACJ is pictured in FIG. 20.

According to one embodiment of the present invention, an agent may beconjugated to an AB. When delivery and release of the agent conjugatedto the AB are desired, immunoglobulin classes that are known to activatecomplement are used. In other applications, carrier immunoglobulins maybe used which are not capable of complement activation. Suchimmunoglobulin carriers may include: certain classes of antibodies suchIgM, IgA, IgD, IgE; certain subclasses of IgG; or certain fragments ofimmunoglobulins, e.g., half ABs (a single heavy: light chain pair), orFab, Fab′ or (Fab′) 2 fragments.

Exemplary AACJs are AAs coupled to a therapeutic agent wherein the AB isdirected to EGFR, CD44, Notch1, 2, 3 or 4 Jagged1 or 2, EpCAM, orIGF-1R.

The chemical linking methods described herein allow the resulting AACJto retain the ability to bind antigen and to activate the complementcascade (when the unconjugated AA also had such ability). As a result,when the AACJ is administered to an individual, the subsequent formationof immune complexes with target antigens in vivo can activate theindividual's serum complement system. The linker is designed to besusceptible to cleavage by complement and so the agent can be cleaved atthe target site by one or more of the enzymes of the complement cascade.The majority of the release of the agent occurs following delivery tothe target site.

In an exemplary embodiment, it is known that all cells of a tumor do noteach possess the target antigenic determinant. Thus, delivery systemswhich require internalization into the target cell will effectsuccessful delivery to those tumor cells that possess the antigenicdeterminant and that are capable of internalizing the conjugate. Tumorcells that do possess the antigenic determinant or are incapable of thisinternalization, will escape treatment. According to the method of thepresent invention, AACJs deliver the agent to the target cells. Moreimportantly, however, once attached to the target cell, the methoddescribed in the present invention allows the release or activation ofthe active or activatable therapeutic agent. Release or activation maybe mediated by the individual's activated by but not limited to thefollowing: complement enzymes, tissue plasminogen activator, urokinase,plasmin or another enzyme having proteolytic activity, or by activationof a photosensitizer or substrate modification. Once released, the agentis then free to permeate the target sites, e.g., tumor mass. As aresult, the agent will act on tumor cells that do not possess theantigenic determinant or could not internalize the conjugate.Additionally, the entire process is not dependent upon internalizationof the conjugate.

(a) Methods for Conjugating Agents

The present invention utilizes several methods for attaching agents toABs (which include antibodies and fragments thereof), two exemplarymethods being attachment to the carbohydrate moieties of the AB, orattachment to sulfhydryl groups of the AB. In certain embodiments, theattachment does not significantly change the essential characteristicsof the AB or the AA itself, such as immunospecificity andimmunoreactivity. Additional considerations include simplicity ofreaction and stability of the antibody conjugate produced. In certainembodiments the AB is first conjugated to one or more agents of interestfollowed by attachment of an MM and CM to produce an AACJ. In otherembodiments the AB is first attached to a MM and CM following which anagent of interest is further conjugated producing an AACJ.

i. Attachment to Oxidized Carbohydrate Moieties

In certain embodiments, agents may be conjugated to the carbohydratemoiety of an AB. Some of the carbohydrate moieties are located on the Fcregion of the immunoglobulin and are required in order for C1 binding tooccur. The carbohydrate moiety of the Fc region of an immunoglobulin maybe utilized in the scheme described herein in the embodiments where theAB is an antibody or antibody fragment that includes at least part of anFc region. Alternatively, the Fab or Fab′ fragments of anyimmunoglobulins which contain carbohydrate moieties may be utilized inthe reaction scheme described herein. An example of such animmunoglobulin is the human IgM sequenced by Putnam et al. (1973,Science 182: 287).

The carbohydrate side chains of antibodies, Fab or Fab′ fragments orother fragments containing an AB may be selectively oxidized to generatealdehydes. A variety of oxidizing agents can be used, such as periodicacid, paraperiodic acid, sodium metaperiodate and potassiummetaperiodate. The resulting aldehydes may then be reacted with aminegroups (e.g., ammonia derivatives such as primary amine, secondaryamine, hydroxylamine, hydrazine, hydrazide, phenylhydrazine,semicarbazide or thiosemicarbazide) to form a Schiff base or reducedSchiff base (e.g., imine, enamine, oxime, hydrazone, phenylhydrazone,semicarbazone, thiosemicarbazone or reduced forms thereof). Chemicalmethods of oxidation of antibodies are provided in U.S. Pat. No.4,867,973 and this patent is incorporated by reference in its entirety.Oxidation of antibodies with these oxidizing agents can be carried outby known methods. In the oxidation, the AB is used generally in the formof an aqueous solution, the concentration being generally less than 100mg/ml, preferably 1 to 20 mg/ml. When an oxygen acid or a salt thereofis used as the oxidizing agent, it is used generally in the form of anaqueous solution, and the concentration is generally 0.001 to 10 mM,sometimes 1.0 to 10 mM. The amount of the oxygen acid or salt thereofdepends on the kind of AB, but generally it is used in excess, forexample, twice to ten times as much as the amount of the oxidizablecarbohydrate. The optimal amount, however, can be determined by routineexperimentation.

In the process for oxidizing ABs with oxygen acids or salts thereof, theoptional ranges include a pH of from about 4 to 8, a temperature of from00 to 37° C., and a reaction period of from about 15 minutes to 12hours. During the oxidation with an oxygen acid or a salt thereof, thereaction can be carried in minimal light to prevent over oxidation.

Alternatively, the carbohydrate moiety of the AB may be modified byenzymatic techniques so as to enable attachment to or reaction withother chemical groups. One example of such an enzyme is galactoseoxidase which oxidizes galactose in the presence of oxygen to form analdehyde. Oxidation of the carbohydrate portion of ABs may also be donewith the enzyme, galactose oxidase (Cooper et al., 1959, J. Biol. Chem.234:445-448). The antibody is used in aqueous solution, theconcentration being generally 0.5 to 20 mg/ml. The enzyme generally isused at about 5 to 100 units per ml of solution, at a pH ranging fromabout 5.5 to about 8.0. The influence of pH, substrate concentration,buffers and buffer concentrations on enzyme reaction are reported inCooper et al., supra.

The AB conjugates, AA conjugates, or AB linker-intermediates of theinvention may be produced by reacting the oxidized AB with any linker oragent having an available amine group selected from the group consistingof primary amine, secondary amine, hydrazine, hydrazide, hydroxylamine,phenylhydrazine, semicarbazide and thiosemicarbazide groups. In anexemplary method, a solution of the oxidized AB or AB linker at aconcentration of from about 0.5 to 20 mg/ml is mixed with the agent orlinker (molar ratios of reactive amine group to antibody aldehyderanging from about 1 to about 10,000) and the solution incubated forfrom about 1 to 18 hours. Suitable temperatures are from 00 to 37° C.and pH may be from about 6 to 8. After the conjugates have been formedthey can optionally be stabilized with a suitable reducing agent, suchas sodium cyanoborohydride or sodium borohydride.

ii. Attachment to Sulfhydryl Groups

When the AB is a full-length antibody or includes at least part of theheavy chain, free sulfhydryl groups can be generated from the disulfidebonds of the immunoglubulin molecule. This is accomplished by mildreduction of the antibody. The disulfide bonds of IgG, which aregenerally susceptible to reduction, are those that link the two heavychains. The disulfide bonds located near the antigen binding region ofthe antibody remain relatively unaffected. Such reduction results in theloss of ability to fix complement but does not interfere withantibody-antigen binding ability (Karush et al, 1979, Biochem. 18:2226-2232). The free sulfhydryl groups generated in the intra-heavychain region can then react with reactive groups of a linker or agent toform a covalent bond which will reduce intereference with the antigenbinding site of the immunoglobulin. Such reactive groups include, butare not limited to, reactive haloalkyl groups (including, for example,haloacetyl groups), p-mercuribenzoate groups and groups capable ofMichael-type addition reactions (including, for example, maleimides andgroups of the type described in Mitra and Lawton, 1979, J. Amer. Chem.Soc. 101: 3097-3110). The haloalkyl can be any alkyl group substitutedwith bromine, iodine or chlorine.

Details of the conditions, methods and materials suitable for mildreduction of antibodies and antibody fragments as described generallyherein may be found in Stanworth and Turner, 1973, In Handbook ofExperimental Immunology, Vol. 1, Second Edition, Weir (ed.), Chapter 10,Blackwell Scientific Publications, London, which chapter is incorporatedherein by reference.

AB-agent conjugates (or AB-linker intermediates) which are produced byattachment to free sulfhydryl groups of reduced immunoglobulin orreduced antibody fragments do not or negligibly activate complement.Thus, these conjugates may be used in in vivo systems where cleavage andrelease of the agent is not desirable (e.g., an enzyme that acts on aspecific substrate). Such conjugates may also be used whennon-complement mediated release is desired. In such an embodiment, theagent may be linked to sulfhydryl groups on the reduced AB via linkerswhich are susceptible to cleavage by enzymes having proteolyticactivity, including but not limited to trypsin, urokinase, plasmin,tissue plasminiogen activator and the like.

Although attachment of an agent to sulfhydryl groups of the AB reducesthe complement fixation ability of the conjugate, such methods ofattachment may be used to make AA conjugates for use in thecomplement-mediated release system. In such an embodiment, an agentjoined to a complement-sensitive substrate linker can be attached tosulfhydryls of reduced ABs or AAs and delivered to the target in amixture with non conjugated AAs that are capable of activatingcomplement. The latter would activate complement which would cleave theagent from the former.

According to one embodiment of the present invention, for attachment tosulfhydryl groups of reduced ABs or AAs, the substrate linkers or theagents are modified by attaching an iodoalkyl group to one end of thelinker. The unmodified site on the linker may or may not be covalentlyattached to an agent. For instance, the substrate linkers which areester or amide linked to agents are modified by the addition of aniodoalkyl group thus forming an iodoalkyl derivative. As mentionedpreviously, the linker may be one that is susceptible or resistant tocleavage by activated complement, trypsin, plasmin, tissue plasminogenactivator, urokinase or another specific enzyme having proteolyticactivity.

(b) Agents for Conjugation to ABs

ABs may be attached to any agent which retains its essential propertiesafter reaction with the AB, and which enables the AB to substantiallyretain immunospecificity and immunoreactivity allowing the AA tofunction as appropriate. The agent can include all chemicalmodifications and derivatives of agents which substantially retain theirbiological activity.

When it is desired to attach an aldehyde of the oxidized carbohydrateportion of an AB to an agent, the agent should contain an amine groupselected from the group consisting of primary amine, secondary amine,hydrazine, hydrazide, hydroxylamine, phenylhydrazine, semicarbazide andthiosemicarbazide groups. If the agent does not contain any such aminogroup, the agent can be modified to introduce a suitable amine groupavailable for coupling.

The agent to be attached to an AB for use in an AA is selected accordingto the purpose of the intended application (i.e, killing, prevention ofcell proliferation, hormone therapy or gene therapy). Such agents mayinclude but is not limited to, for example, pharmaceutical agents,toxins, fragments of toxins, alkylating agents, enzymes, antibiotics,antimetabolites, antiproliferative agents, hormones, neurotransmitters,DNA, RNA, siRNA, oligonucleotides, antisense RNA, aptamers, diagnostics,radioopaque dyes, radioactive isotopes, fluorogenic compounds, magneticlabels, nanoparticles, marker compounds, lectins, compounds which altercell membrane permeability, photochemical compounds, small molecules,liposomes, micelles, gene therapy vectors, viral vectors, and the like.Non-limiting Table 4 lists some of the exemplary pharmaceutical agentsthat may be employed in the herein described invention but in no way ismeant to be an exhaustive list. Finally, combinations of agents orcombinations of different classes of agents may be used.

According to one embodiment of the present invention, photochemicalsincluding photosensitizers and photothermolytic agents may be used asagents. Efficient photosensitizers include, but are not limited toporphyrins and modified porphyrins (e.g., hematoporphyrin,hematoporphyrin dihyddrazide, deuteroporphyrin dihydrazide andprotoporphyrin dihydrazide), rose bengal, acridines, thiazines,xanthenes, anthraquinones, azines, flavin and nonmetal-containingporphyrins, porphyrin-like compounds, methylene blue, eosin, psoralinand the like. Other photosensitizers include, but are not limited totetracyclines (e.g., dimethylchlor tetracycline) sulfonamides (e.g.,sulfanilamide), griseofulvin, phenothiazines, (e.g., chlorpromazine),thiazides, sulfonylurea, and many others. Photochemicals may be designedor synthetically prepared to absorb light at specific wavelengths.Photothermolytic agents, such as Azure A, which are activated at thesite of action by a light source (see Anderson and Parrish, 1983,Science 220: 524-527) may be utilized as agents.

According to another embodiment of the present invention, enzymes thatcatalyze substrate modification with the production of cytotoxicby-products may be used as agents. Examples of such enzymes include butare not limited to glucose oxidase, galactose oxidase, xanthene oxidaseand the like.

TABLE 4 Exemplary Pharmaceutical Agents for Conjugation NAME/CLASSLINKAGE MANUFACTURERS(S) I. ANTIBACTERIALS Aminoglycosides Streptomycinester/amide Neomycin ester/amide Dow, Lilly, Dome, Pfipharmics Kanamycinester/amide Bristol Amikacin ester Bristol Gentamicin ester/amideUpjohn, Wyeth, Schering Tobramycin ester/amide Lilly Streptomycin Bester/amide Squibb Spectinomycin ester Upjohn Ampicillin amide Squibb,Parke-Davis, Comer, Wyeth, Upjohn, Bristol, SKF Sulfanilamide amideMerrell-National Burroughs-Wellcome, Dow, Polymyxin amide Parke-DavisChloramphenicol ester Parke-Davis II. ANTIVIRALS AcyclovirBurroughs-Wellcome Vira A ester/amide Parke-Davis Symmetrel amide EndoIII. ANTIFUNGALS Nystatin ester Squibb, Primo, Lederle, Pfizer,Holland-Rantor IV. ANTINEOPLASTICS Adriamycin ester/amide AdriaCerubidine ester/amide Ives Bleomycin ester/amide Bristol Alkeran amideBurroughs-Wellcome Velban ester Lilly Oncovin ester Lilly Fluorouracilester Adria, Roche, Herbert Methotrexate amide Lederle Thiotepa —Lederle Bisantrene — Lederle Novantrone ester Lederle Thioguanine amideBurroughs-Wellcome Procarabizine — Hoffman La Roche Cytarabine — UpjohnV. RADIO-PHARMACEUTICALS ¹²⁵I ¹³¹I ^(99m)Tc (Technetium) VI. HEAVYMETALS Barium Gold Platinum VII. ANTIMYCOPLASMALS Tylosine Spectinomycin

(c) Linkers for Conjugating Agents

The present invention utilizes several methods for attaching agents toABs: (a) attachment to the carbohydrate moieties of the AB, or (b)attachment to sulfhydryl groups of the AB. According to the invention,ABs may be covalently attached to an agent through an intermediatelinker having at least two reactive groups, one to react with AB and oneto react with the agent. The linker, which may include any compatibleorganic compound, can be chosen such that the reaction with AB (oragent) does not adversely affect AB reactivity and selectivity.Furthermore, the attachment of linker to agent might not destroy theactivity of the agent. Suitable linkers for reaction with oxidizedantibodies or oxidized antibody fragments include those containing anamine selected from the group consisting of primary amine, secondaryamine, hydrazine, hydrazide, hydroxylamine, phenylhydrazine,semicarbazide and thiosemicarbazide groups. Such reactive functionalgroups may exist as part of the structure of the linker, or may beintroduced by suitable chemical modification of linkers not containingsuch groups.

According to the present invention, suitable linkers for attachment toreduced ABs include those having certain reactive groups capable ofreaction with a sulfhydryl group of a reduced antibody or fragment. Suchreactive groups include, but are not limited to: reactive haloalkylgroups (including, for example, haloacetyl groups), p-mercuribenzoategroups and groups capable of Michael-type addition reactions (including,for example, maleimides and groups of the type described by Mitra andLawton, 1979, J. Amer. Chem. Soc. 101: 3097-3110).

The agent may be attached to the linker before or after the linker isattached to the AB. In certain applications it may be desirable to firstproduce an AB-linker intermediate in which the linker is free of anassociated agent. Depending upon the particular application, a specificagent may then be covalently attached to the linker. In otherembodiments the AB is first attached to the MM, CM and associatedlinkers and then attached to the linker for conjugation purposes.

(i) Branched Linkers:

In specific embodiments, branched linkers which have multiple sites forattachment of agents are utilized. For multiple site linkers, a singlecovalent attachment to an AB would result in an AB-linker intermediatecapable of binding an agent at a number of sites. The sites may bealdehyde or sulfhydryl groups or any chemical site to which agents canbe attached. Alternatively, higher specific activity (or higher ratio ofagents to AB) can be achieved by attachment of a single site linker at aplurality of sites on the AB. This plurality of sites may be introducedinto the AB by either of two methods. First, one may generate multiplealdehyde groups and/or sulfhydryl groups in the same AB. Second, one mayattach to an aldehyde or sulfhydryl of the AB a “branched linker” havingmultiple functional sites for subsequent attachment to linkers. Thefunctional sites of the branched linker or multiple site linker may bealdehyde or sulfhydryl groups, or may be any chemical site to whichlinkers may be attached. Still higher specific activities may beobtained by combining these two approaches, that is, attaching multiplesite linkers at several sites on the AB.

(ii) Cleavable Linkers:

Peptide linkers which are susceptible to cleavage by enzymes of thecomplement system, such as but not limited to urokinase, tissueplasminogen activator, trypsin, plasmin, or another enzyme havingproteolytic activity may be used in one embodiment of the presentinvention. According to one method of the present invention, an agent isattached via a linker susceptible to cleavage by complement. Theantibody is selected from a class which can activate complement. Theantibody-agent conjugate, thus, activates the complement cascade andreleases the agent at the target site. According to another method ofthe present invention, an agent is attached via a linker susceptible tocleavage by enzymes having a proteolytic activity such as a urokinase, atissue plasimogen activator, plasmin, or trypsin. Non-liming examples ofcleavable linker sequences are provided in Table 5.

TABLE 5 Exemplary Linker Sequences for Conjugation Types of CleavableSequences Amino Acid Sequence Plasmin cleavable sequences Pro-urokinasePRFKIIGG (SEQ ID NO: 20) PRFRIIGG (SEQ ID NO: 21) TGFβ SSRHRRALD (SEQ IDNO: 22) Plasminogen RKSSIIIRMRDVVL (SEQ ID NO: 23) StaphylokinaseSSSFDKGKYKKGDDA (SEQ ID NO: 24) SSSFDKGKYKRGDDA (SEQ ID NO: 25) FactorXa cleavable IEGR (SEQ ID NO: 26) sequences IDGR (SEQ ID NO: 27) GGSIDGR(SEQ ID NO: 28) MMP cleavable sequences Gelatinase A PLGLWA (SEQ ID NO:29) Collagenase cleavable sequences Calf skin collagen GPQGIAGQ (SEQ IDNO: 30) (α1(I) chain) Calf skin collagen GPQGLLGA (SEQ ID NO: 31) (α2(I)chain) Bovine cartilage GIAGQ (SEQ ID NO: 32) collagen (α1(II) chain)Human liver GPLGIAGI (SEQ ID NO: 33) collagen (α1(III) chain) Human α₂MGPEGLRVG (SEQ ID NO: 34) Human PZP YGAGLGVV (SEQ ID NO: 35) AGLGVVER(SEQ ID NO: 36) AGLGISST (SEQ ID NO: 37) Rat α₁M EPQALAMS (SEQ ID NO:38) QALAMSAI (SEQ ID NO: 39) Rat α₂M AAYHLVSQ (SEQ ID NO: 40) MDAFLESS(SEQ ID NO: 41) Rat α₁I₃(2J) ESLPVVAV (SEQ ID NO: 42) Rat α₁I₃(27J)SAPAVESE (SEQ ID NO: 43) Human fibroblast DVAQFVLT (SEQ ID NO: 44)collagenase VAQFVLTE (SEQ ID NO: 45) (autolytic AQFVLTEG (SEQ ID NO: 46)cleavages) PVQPIGPQ (SEQ ID NO: 47)

In addition agents may be attached via disulfide bonds (for example, thedisulfide bonds on a cysteine molecule) to the AB. Since many tumorsnaturally release high levels of glutathione (a reducing agent) this canreduce the disulfide bonds with subsequent release of the agent at thesite of delivery. In certain specific embodiments the reducing agentthat would modify a CM would also modify the linker of the conjugatedAA.

(iii) Spacers and Cleavable Elements.

In still another embodiment, it may be necessary to construct the linkerin such a way as to optimize the spacing between the agent and the AB ofthe AA. This may be accomplished by use of a linker of the generalstructure:W—(CH2)n-QwhereinW is either —NH—CH2- or —CH2-;Q is an amino acid, peptide; andn is an integer from 0 to 20.

In still other embodiments, the linker may comprise a spacer element anda cleavable element. The spacer element serves to position the cleavableelement away from the core of the AB such that the cleavable element ismore accessible to the enzyme responsible for cleavage. Certain of thebranched linkers described above may serve as spacer elements.

Throughout this discussion, it should be understood that the attachmentof linker to agent (or of spacer element to cleavable element, orcleavable element to agent) need not be particular mode of attachment orreaction. Any reaction providing a product of suitable stability andbiological compatibility is acceptable.

(iv) Serum Complement and Selection of Linkers:

According to one method of the present invention, when release of anagent is desired, an AB that is an antibody of a class which canactivate complement is used. The resulting conjugate retains both theability to bind antigen and activate the complement cascade. Thus,according to this embodiment of the present invention, an agent isjoined to one end of the cleavable linker or cleavable element and theother end of the linker group is attached to a specific site on the AB.For example, if the agent has an hydroxy group or an amino group, it maybe attached to the carboxy terminus of a peptide, amino acid or othersuitably chosen linker via an ester or amide bond, respectively. Forexample, such agents may be attached to the linker peptide via acarbodimide reaction. If the agent contains functional groups that wouldinterfere with attachment to the linker, these interfering functionalgroups can be blocked before attachment and deblocked once the productconjugate or intermediate is made. The opposite or amino terminus of thelinker is then used either directly or after further modification forbinding to an AB which is capable of activating complement.

Linkers (or spacer elements of linkers) may be of any desired length,one end of which can be covalently attached to specific sites on the ABof the AA. The other end of the linker or spacer element may be attachedto an amino acid or peptide linker.

Thus when these conjugates bind to antigen in the presence of complementthe amide or ester bond which attaches the agent to the linker will becleaved, resulting in release of the agent in its active form. Theseconjugates, when administered to a subject, will accomplish delivery andrelease of the agent at the target site, and are particularly effectivefor the in vivo delivery of pharmaceutical agents, antibiotics,antimetabolites, antiproliferative agents and the like as presented inbut not limited to those in Table 4.

(v) Linkers for Release without Complement Activation:

In yet another application of targeted delivery, release of the agentwithout complement activation is desired since activation of thecomplement cascade will ultimately lyse the target cell. Hence, thisapproach is useful when delivery and release of the agent should beaccomplished without killing the target cell. Such is the goal whendelivery of cell mediators such as hormones, enzymes, corticosteroids,neurotransmitters, genes or enzymes to target cells is desired. Theseconjugates may be prepared by attaching the agent to an AB that is notcapable of activating complement via a linker that is mildly susceptibleto cleavage by serum proteases. When this conjugate is administered toan individual, antigen-antibody complexes will form quickly whereascleavage of the agent will occur slowly, thus resulting in release ofthe compound at the target site.

(vi) Biochemical Cross Linkers:

In other embodiments, the AA may be conjugated to one or moretherapeutic agents using certain biochemical cross-linkers.Cross-linking reagents form molecular bridges that tie togetherfunctional groups of two different molecules. To link two differentproteins in a stepwise manner, hetero-bifunctional cross-linkers can beused that eliminate unwanted homopolymer formation. Exemplaryhetero-bifunctional cross-linkers are referenced in Table 6.

TABLE 6 Exemplary Hetero-Bifunctional Cross Linkers HETERO-BIFUNCTIONALCROSS-LINKERS Spacer Arm Length Linker Reactive Toward Advantages andApplications after cross-linking SMPT Primary amines Greater stability11.2 A Sulfhydryls SPDP Primary amines Thiolation 6.8 A SulfhydrylsCleavable cross-linking LC-SPDP Primary amines Extended spacer arm 15.6A Sulfhydryls Sulfo-LC-SPDP Primary amines Extender spacer arm 15.6 ASulfhydryls Water-soluble SMCC Primary amines Stable malcimide reactivegroup 11.6 A Sulfhydryls Enzyme-antibody conjugation Hapten-carrierprotein conjugation Sulfo-SMCC Primary amines Stable maleimide reactivegroup 11.6 A Sulfhydryls Water-soluble Enzyme-antibody conjugation MBSPrimary amines Enzyme-antibody conjugation 9.9 A SulfhydrylsHapten-carrier protein conjugation Sulfo-MBS Primary aminesWater-soluble 9.9 A Sulfhydryls SIAB Primary amines Enzyme-antibodyconjugation 10.6 A Sulfhydryls Sulfo-SIAB Primary amines Water-soluble10.6 A Sulfhydryls SMPB Primary amines Extended spacer arm 14.5 ASulfhydryls Enzyme-antibody conjugation Sulfo-SMPB Primary aminesExtended spacer arm 14.5 A Sulfhydryls Water-soluble EDE/Sulfo-NHSPrimary amines Hapten-Carrier conjugation 0 Carboxyl groups ABHCarbohydrates Reacts with sugar groups 11.9 A Nonselective

(vii) Non-Cleavable Linkers or Direct Attachment:

In still other embodiments of the invention, the conjugate may bedesigned so that the agent is delivered to the target but not released.This may be accomplished by attaching an agent to an AB either directlyor via a non-cleavable linker.

These non-cleavable linkers may include amino acids, peptides, D-aminoacids or other organic compounds which may be modified to includefunctional groups that can subsequently be utilized in attachment to ABsby the methods described herein. A—general formula for such an organiclinker could beW—(CH2)n-Qwherein W is either —NH—CH2- or —CH2-;Q is an amino acid, peptide; andn is an integer from 0 to 20.

(viii) Non-Cleavable Conjugates.

Alternatively, a compound may be attached to ABs which do not activatecomplement. When using ABs that are incapable of complement activation,this attachment may be accomplished using linkers that are susceptibleto cleavage by activated complement or using linkers that are notsusceptible to cleavage by activated complement.

(d) Uses of Activatable Antibody Conjugates

The AA-agent conjugates (AACJs) of the invention are useful intherapeutics, diagnostics, substrate modification and the like.

The AACJs of the invention are useful in a variety of therapeutic invivo applications such as but not limited to the treatment of neoplasms,including cancers, adenomas, and hyperplasias; certain immunologicaldisorders, including autoimmune diseases, graft-versus-host diseases(e.g., after bone marrow transplantation), immune suppressive diseases,e.g., after kidney or bone marrow transplantation. Treatment of suchcellular disorders involving, for example, bone marrow transplantation,may include purging (by killing) undesired cells, e.g., malignant cellsor mature T lymphocytes.

Therapeutic applications center generally on treatment of variouscellular disorders, including those broadly described above, byadministering an effective amount of the antibody-agent conjugates ofthe invention. The properties of the antibody are such that it isimmunospecific for and immunoreactive with a particular antigen renderit ideally suited for delivery of agents to specific cells, tissues,organs or any other site having that particular antigen.

According to this aspect of the invention, the AACJ functions to deliverthe conjugate to the target site.

The choice of ABs, linkers, and agents used to make the AACJs dependsupon the purpose of delivery. The delivery and release or activation ofagents at specific target sites may result in selective killing orinhibition of proliferation of tumor cells, cancer cells, fungi,bacteria, parasites, or virus. The targeted delivery of hormones,enzymes, or neurotransmitters to selected sites may also beaccomplished. Ultimately the method of the present invention may have anapplication in gene therapy programs wherein DNA or specific genes maybe delivered in vivo or in vitro to target cells that are deficient inthat particular gene. Additionally, the conjugates may be used to reduceor prevent the activation of oncogenes, such as myc, ras and the like.

In vivo administration may involve use of agents of AACJs in anysuitable adjuvant including serum or physiological saline, with orwithout another protein, such as human serum albumin. Dosage of theconjugates may readily be determined by one of ordinary skill, and maydiffer depending upon the nature of the cellular disorder and the agentused. Route of administration may be parenteral, with intravenousadministration generally preferred.

(i) Substrate Modification

In an alternate embodiment of the present invention, substrateactivation by the agent may be used to mediate formation of singletoxygen or peroxides and induce cell killing. In this particularembodiment, the agent is an enzyme. For example, galactose oxidase willoxidize galactose and some galactose derivatives at the C 6 position. Inthe course of the oxidation reaction, molecular oxygen is converted intohydrogen peroxide which is toxic to neighboring cells. The enzymeglucose oxidase, a flavoenzyme, may also be used in the embodiment ofthis invention. This enzyme is highly specific for β-D-glucose and canact as an antibiotic due to peroxide formation. The enzyme may beattached to an AB either directly or via a non-cleavable linker. Asubject is given an effective dosage of this AACJ and is then perfusedwith substrate. Cell killing is mediated through the formation ofperoxides by the methods described above. The toxic effect of peroxidesmay be amplified by administration of a second enzyme, preferably ofhuman origin, to convert its peroxide to a more toxic hypochlorous acid.Examples of suitable enzymes include but are not limited tomyeloperoxidase, lactoperoxidase and chloroperoxidase.

Display Methods and Compositions for Identifying and/or Optimizing AAs

Methods for identifying and optimizing AAs, as well as compositionsuseful in such methods, are described below.

(a) Libraries of AAs or Candidate AAs Displayed on Replicable BiologicalEntities

In general, the screening methods to identify an AA and/or to optimizean AA for a switchable phenotype can involve production of a library ofreplicable biological entities that display on their surface a pluralityof different candidate AAs. These libraries can then be subjected toscreening methods to identify candidate AAs having one or more desiredcharacteristics of an AA.

The candidate AA libraries can contain candidate AAs that differ by oneor more of the MM, linker (which may be part of the MM), CM (which maybe part of the MM), and AB. In one embodiment the AAs in the library arevariable for the MM and/or the linker, with the AB and CM beingpreselected. Where the AA is to include pairs of cysteine residues toprovide a disulfide bond in the AA, the relative position of thecysteines in the AA can be varied.

The library for screening is generally provided as a library ofreplicable biological entities which display on their surface differentcandidate AAs. For example, a library of candidate AAs can include aplurality of candidate AAs displayed on the surface of population of areplicable biological entities, wherein each member of said plurality ofcandidate AAs comprises: (a) an antibody or fragment thereof (AB); (b) acleavable moiety (CM); and (c) a candidate masking moiety (candidateMM), wherein the AB, CM and candidate MM are positioned such that theability of the candidate MM to inhibit binding of the AB to a target inan uncleaved state and allow binding of the AB to the target in acleaved state can be determined. Suitable replicable biological entitiesinclude cells (e.g., bacteria (e.g., E. coli), yeast (e.g., S.cerevesiae), protozoan cells, mammalian cells), bacteriophage, andviruses. Antibody display technologies are well known in the art.

(b) Display of Candidate AAs on the Surface of Replicable BiologicalEntities

A variety of display technologies using replicable biological entitiesare known in the art. These methods and entities include, but are notlimited to, display methodologies such as mRNA and ribosome display,eukaryotic virus display, and bacterial, yeast, and mammalian cellsurface display. See Wilson, D. S., et al. 2001 PNAS USA98(7):3750-3755; Muller, O. J., et al. (2003) Nat. Biotechnol. 3:312;Bupp, K. and M. J. Roth (2002) Mol. Ther. 5(3):329 3513; Georgiou, G.,et al., (1997) Nat. Biotechnol. 15(1):29 3414; and Boder, E. T. and K.D. Wittrup (1997) Nature Biotech. 15(6):553 557. Surface display methodsare attractive since they enable application of fluorescence-activatedcell sorting (FACS) for library analysis and screening. See Daugherty,P. S., et al. (2000) J. Immunol. Methods 243(1 2):211 2716; Georgiou, G.(2000) Adv. Protein Chem. 55:293 315; Daugherty, P. S., et al. (2000)PNAS USA 97(5):2029 3418; Olsen, M. J., et al. (2003) Methods Mol. Biol.230:329 342; Boder, E. T. et al. (2000) PNAS USA 97(20):10701 10705;Mattheakis, L. C., et al. (1994) PNAS USA 91(19): 9022 9026; and Shusta,E. V., et al. (1999) Curr. Opin. Biotech. 10(2):117 122. Additionaldisplay methodologies which may be used to identify a peptide capable ofbinding to a biological target of interest are described in U.S. Pat.No. 7,256,038, the disclosure of which is incorporated herein byreference.

A display scaffold refers to a polypeptide which when expressed in ahost cell is presented on an extracellularly accessible surface of thehost cell and provides for presentation of an operably linkedheterologous polypeptide. For example, display scaffolds find use in themethods disclosed herein to facilitate screening of candidate AAs.Display scaffolds can be provided such that a heterologous polypeptideof interest can be readily released from the display scaffold, e.g. byaction of a protease that facilitates cleavage of the fusion protein andrelease of a candidate AA from the display scaffold.

Phage display involves the localization of peptides as terminal fusionsto the coat proteins, e.g., pIII, pIIV of bacteriophage particles. SeeScott, J. K. and G. P. Smith (1990) Science 249(4967):386 390; andLowman, H. B., et al. (1991) Biochem. 30(45):10832 10838. Generally,polypeptides with a specific function of binding are isolated byincubating with a target, washing away non-binding phage, eluting thebound phage, and then re-amplifying the phage population by infecting afresh culture of bacteria.

Exemplary phage display and cell display compositions and methods aredescribed in U.S. Pat. Nos. 5,223,409; 5,403,484; 7,118,159; 6,979,538;7,208,293; 5,571,698; and 5,837,500.

Additional exemplary display scaffolds and methods include thosedescribed in U.S. Patent Application Publication No: 2007/0065158,published Mar. 22, 2007.

Optionally, the display scaffold can include a protease cleavage site(different from the protease cleavage site of the CM) to allow forcleavage of an AA or candidate AA from a surface of a host cell.

In one embodiment, where the replicable biological entity is a bacterialcell, suitable display scaffolds include circularly permuted Escherichiacoli outer membrane protein OmpX (CPX) described by Rice et al, ProteinSci. (2006) 15: 825-836. See also, U.S. Pat. No. 7,256,038, issued Aug.14, 2007.

(c) Constructs Encoding AAs

The disclosure further provides nucleic acid constructs which includesequences coding for AAs and/or candidate AAs. Suitable nucleic acidconstructs include, but are not limited to, constructs which are capableof expression in a prokaryotic or eukaryotic cell. Expression constructsare generally selected so as to be compatible with the host cell inwhich they are to be used.

For example, non-viral and/or viral constructs vectors may be preparedand used, including plasmids, which provide for replication of an AA- orcandidate AA-encoding DNA and/or expression in a host cell. The choiceof vector will depend on the type of cell in which propagation isdesired and the purpose of propagation. Certain constructs are usefulfor amplifying and making large amounts of the desired DNA sequence.Other vectors are suitable for expression in cells in culture. Thechoice of appropriate vector is well within the skill of the art. Manysuch vectors are available commercially. Methods for generatingconstructs can be accomplished using methods well known in the art.

In order to effect expression in a host cell, the polynucleotideencoding an AA or candidate AA is operably linked to a regulatorysequence as appropriate to facilitate the desired expression properties.These regulatory sequences can include promoters, enhancers,terminators, operators, repressors, silencers, inducers, and 3′ or 5′UTRs. Expression constructs generally also provide a transcriptional andtranslational initiation region as may be needed or desired, which maybe inducible or constitutive, where the coding region is operably linkedunder the transcriptional control of the transcriptional initiationregion, and a transcriptional and translational termination region.These control regions may be native to the species from which thenucleic acid is obtained, or may be derived from exogenous sources.

Promoters may be either constitutive or regulatable. In some situationsit may be desirable to use conditionally active promoters, such asinducible promoters, e.g., temperature-sensitive promoters. Inducibleelements are DNA sequence elements that act in conjunction withpromoters and may bind either repressors (e.g. lacO/LAC Iq repressorsystem in E. coli) or inducers (e.g. gal1/GAL4 inducer system in yeast).In such cases, transcription is virtually shut off until the promoter isde-repressed or induced, at which point transcription is turned-on.

Constructs, including expression constructs, can also include aselectable marker operative in the host to facilitate, for example,growth of host cells containing the construct of interest. Suchselectable marker genes can provide a phenotypic trait for selection oftransformed host cells such as dihydrofolate reductase or neomycinresistance for eukaryotic cell culture.

Expression constructs can include convenient restriction sites toprovide for the insertion and removal of nucleic acid sequences encodingthe AA and/or candidate AA. Alternatively or in addition, the expressionconstructs can include flanking sequences that can serve as the basisfor primers to facilitate nucleic acid amplification (e.g., PCR-basedamplification) of an AA-coding sequence of interest.

The above described expression systems may be employed with prokaryotesor eukaryotes in accordance with conventional ways, depending upon thepurpose for expression. In some embodiments, a unicellular organism,such as E. coli, B. subtilis, S. cerevisiae, insect cells in combinationwith baculovirus vectors, or cells of a higher organism such asvertebrates, e.g. COS 7 cells, HEK 293, CHO, Xenopus Oocytes, etc., maybe used as the expression host cells. Expression systems for each ofthese classes and types of host cells are known in the art.

(d) Methods of Making Libraries of AAs/Candidate AAs Displayed onReplicable Biological Entities

The present disclosure contemplates methods of making the libraries ofAAs and/or candidate AAs described herein.

In one embodiment, a method of making an AA library and/or candidate AAlibrary comprises: (a) constructing a set of recombinant DNA vectors asdescribed herein that encode a plurality of AAs and/or candidate AAs;(b) transforming host cells with the vectors of step (a); and (c)culturing the host cells transformed in step (b) under conditionssuitable for expression and display of the fusion polypeptides.

(e) Production of Nucleic Acid Sequences Encoding Candidate AAs

Production of candidate AAs for use in the screening methods can beaccomplished using methods known in the art. Polypeptide display, singlechain antibody display, antibody display and antibody fragment displayare methods well known in the art. In general, an element of an AA e.g.,MM, to be varied in the candidate AA library is selected forrandomization. The candidate AAs in the library can be fully randomizedor biased in their randomization, e.g. in nucleotide/residue frequencygenerally or in position of amino acid(s) within an element.

Methods of Screening for AAs

The present disclosure provides methods of identifying AAs, which can beenzymatically activated AAs, reducing agent-susceptible AAs, or an AAthat is activatable by either or both of enzymatic activation orreducing agent-based activation. Generally, the methods includecontacting a plurality of candidate AAs with a target capable of bindingan AB of the AAs and a protease capable of cleaving a CM of the AAs,selecting a first population of members of said plurality which bind tothe target when exposed to protease, contacting said first populationwith the target in the absence of the protease, and selecting a secondpopulation of members from said first population by depleting from saidfirst population members that bind the target in the absence of theprotease, wherein said method provides for selection of candidate AAswhich exhibit decreased binding to the target in the absence of theprotease as compared to target binding in the presence of the protease.

In general, the method for screening for candidate AAs having a desiredswitchable phenotype is accomplished through a positive screening step(to identify members that bind target following exposure to protease)and a negative screening step (to identify members that do not bindtarget when not exposed to protease). The negative screening step can beaccomplished by, for example, depleting from the population members thatbind the target in the absence of the protease. It should be noted thatthe library screening methods described herein can be initiated byconducting the negative screening first to select for candidates that donot bind labeled target in the absence of enzyme treatment (i.e., do notbind labeled target when not cleaved), and then conducting the positivescreening (i.e., treating with enzyme and selecting for members whichbind labeled target in the cleaved state). However, for convenience, thescreening method is described below with the positive selection as afirst step.

The positive and negative screening steps can be conveniently conductedusing flow cytometry to sort candidate AAs based on binding of adetectably labeled target. One round or cycle of the screening procedureinvolves both a positive selection step and a negative selection step.The methods may be repeated for a library such that multiple cycles(including complete and partial cycles, e.g., 1.5 cycles, 2.5 cycles,etc.) are performed. In this manner, members of the plurality ofcandidate AAs that exhibit the switching characteristics of an AA may beenriched in the resulting population.

In general, the screening methods are conducted by first generating anucleic acid library encoding a plurality of candidate AAs in a displayscaffold, which is in turn introduced into a display scaffold forexpression on the surface of a replicable biological entity. As usedherein, a plurality of candidate AAs refers to a plurality ofpolypeptides having amino acid sequences encoding candidate AAs, wheremembers of the plurality are variable with respect to the amino acidsequence of at least one of the components of an AA, e.g., the pluralityis variable with respect to the amino acid sequence of the MM, the CM orthe AB, usually the MM.

For example, the AB and CM of the candidate AAs are held fixed and thecandidate AAs in the library are variable with respect to the amino acidsequence of the MM. In another example, a library can be generated toinclude candidate AAs having an MM that is designed to position acysteine residue such that disulfide bond formation with anothercysteine in the candidate AA is favored (with other residues selected toprovide an MM having an amino acid sequence that is otherwise fully orat least partially randomized). In another example, a library can begenerated to include candidate AAs in which the MM includes a fullyrandomized amino acid sequence. Such libraries can contain candidate AAsdesigned by one or more of these criterion. By screening members of saidplurality according to the methods described herein, members havingcandidate MMs that provide a desired switchable phenotype can beidentified.

In one embodiment of the methods, each member of the plurality ofcandidate AAs is displayed on the surface of a replicable biologicalentity (exemplified here by bacterial cells). The members of theplurality are exposed to a protease capable of cleaving the CM of thecandidate AAs and contacted with a target which is a binding partner ofthe AB of the candidate AAs. Bacterial cells displaying memberscomprising ABs which bind the target after exposure to the protease areidentified and/or separated via detection of target binding (e.g.,detection of a target-AB complex). Members comprising ABs which bind thetarget after protease exposure (which can lead to cleavage of the CM)are then contacted with the target in the absence of the protease.Bacterial cells displaying members comprising ABs which exhibitdecreased or undetectable binding to the target in the absence ofcleavage are identified and/or separated via detection of cells lackingbound target. In this manner, members of the plurality of candidate AAswhich bind target in a cleaved state and exhibit decreased orundetectable target binding in an uncleaved state are identified and/orselected.

As noted above, candidate AA libraries can be constructed so as toscreen for one or more aspects of the AA constructs, e.g., to providefor optimization of a switchable phenotype for one or more of the MM,the CM, and the AB. One or more other elements of the AA can be variedto facilitate optimization. For example: vary the MM, including varyingthe number or position of cysteines or other residues that can providefor different conformational characteristics of the AA in the absence ofcleaving agent (e.g., enzyme): vary the CM to identify a substrate thatis optimized for one or more desired characteristics (e.g., specificityof enzyme cleavage, and the like); and/or vary the AB to provide foroptimization of switchable target binding.

In general, the elements of the candidate AA libraries are selectedaccording to a target protein of interest, where the AA is to beactivated to provide for enhanced binding of the target in the presenceof a cleaving agent (e.g., enzyme) that cleaves the CM. For example,where the CM and AB are held fixed among the library members, the CM isselected such that it is cleavable by a cleaving agent (e.g., enzyme)that is co-localized with a target of interest, where the target ofinterest is a binding partner of the AB. In this manner, an AA can beselected such that it is selectively activated under the appropriatebiological conditions, and thus at an appropriate biological location.For example, where it is desired to develop an AA to be used as ananti-angiogenic compound and exhibit a switchable phenotype for VEGFbinding, the CM of the candidate AA is selected to be a substrate for anenzyme and/or a reducing agent that is co-localized with VEGF (e.g., aCM cleavable by a matrix-metalloprotease). By way of another example,where it is desired to develop an AA to be used as an anti-angiogeniccompound and exhibit a switchable phenotype for Notch receptor binding,Jagged ligand binding, or EGFR binding, the CM of the candidate AA isselected to be a substrate for an enzyme and/or a reducing agent that isco-localized with the Notch receptor, Jagged ligand, or EGFR (e.g., a CMcleavable by a uPA or plasmin).

As discussed above, an AB is generally selected according to a target ofinterest. Many targets are known in the art. Biological targets ofinterest include protein targets that have been identified as playing arole in disease. Such targets include but are not limited to cellsurface receptors and secreted binding proteins (e.g., growth factors),soluble enzymes, structural proteins (e.g. collagen, fibronectin),intracellular targets, and the like. Exemplary non-limiting targets arepresented in Table 1, but other suitable targets will be readilyidentifiable by those of ordinary skill in the art. In addition, manyproteases are known in the art which co-localize with targets ofinterest. As such, persons of ordinary skill in the art will be able toreadily identify appropriate enzymes and enzyme substrates for use inthe above methods.

(a) Optional Enrichment for Cell Surface Display Prior to AA Screening

Prior to the screening method, it may be desirable to enrich for cellsexpressing an appropriate peptide display scaffold on the cell surface.The optional enrichment allows for removal of cells from the celllibrary that (1) do not express peptide display scaffolds on the cellouter membrane or (2) express non-functional peptide display scaffoldson the cell outer membrane. A non-functional peptide display scaffolddoes not properly display a candidate AA, e.g., as a result of a stopcodon or a deletion mutation.

Enrichment for cells can be accomplished by growing the cell populationand inducing expression of the peptide display scaffolds. The cells arethen sorted based on, for example, detection of a detectable signal ormoiety incorporated into the scaffold or by use of a detectably-labeledantibody that binds to a shared portion of the display scaffold or theAA. These methods are described in greater detail in U.S. PatentApplication Publication No: 2007/0065158, published Mar. 22, 2007.

(b) Screening for Target Binding by Cleaved AAs

Prior to screening, the candidate AA library can be expanded (e.g., bygrowth in a suitable medium in culture under suitable conditions).Subsequent to the optional expansion, or as an initial step, the libraryis subjected to a first screen to identify candidate AAs that bindtarget following exposure to protease. Accordingly, this step is oftenreferred to herein as the positive selection step.

In order to identify members that bind target following proteasecleavage, the candidate AA library is contacted with a protease capableof cleaving the CM of the displayed candidate AAs for an amount of timesufficient and under conditions suitable to provide for cleavage of theprotease substrate of the CM. A variety of protease-CM combinations willbe readily ascertainable by those of ordinary skill in the art, wherethe protease is one which is capable of cleaving the CM and one whichco-localizes in vivo with a target of interest (which is a bindingpartner of the AB). For example, where the target of interest is a solidtumor associated target (e.g. VEGF), suitable enzymes include, forexample, Matrix-Metalloproteases (e.g., MMP-2), A Disintegrin andMetalloprotease(s) (ADAMs)/ADAM with thrombospondin-like motifs(ADAMTS), Cathepsins and Kallikreins. The amino acid sequences ofsubstrates useful as CMs in the AAs described herein are known in theart and, where desired, can be screened to identify optimal sequencessuitable for use as a CM by adaptation of the methods described herein.Exemplary substrates can include but are not limited to substratescleavable by enzymes listed in Table 3.

The candidate AA library is also exposed to target for an amount of timesufficient and under conditions suitable for target binding, whichconditions can be selected according to conditions under which targetbinding to the AB would be expected. The candidate AA library can beexposed to the protease prior to exposure to target (e.g., to provide apopulation of candidate AAs which include cleaved AAs) or in combinationwith exposure to target, usually the latter so as to best model theexpected in vivo situation in which both protease and target will bepresent in the same environmental milieu. Following exposure to bothprotease and target, the library is then screened to select membershaving bound target, which include candidate AAs in a target-AB complex.

Detection of target-bound candidate AAs can be accomplished in a varietyof ways. For example, the target may be detectably labeled and the firstpopulation of target-bound candidate AAs may be selected by detection ofthe detectable label to generate a second population having bound target(e.g., a positive selection for target-bound candidate AAs).

(c) Screening for Candidate AAs that do not Bind Target in the Absenceof Protease Cleavage

The population of candidate AAs selected for target binding followingexposure to protease can then be expanded (e.g., by growth in a suitablemedium in culture under suitable conditions), and the expanded librarysubjected to a second screen to identify members exhibiting decreased orno detectable binding to target in the absence of protease exposure. Thepopulation resulting from this second screen will include candidate AAsthat, when uncleaved, do not bind target significantly or to adetectable level. Accordingly, this step is often referred to herein asthe negative selection step.

The population that resulted from the first screen is contacted withtarget in the absence of the protease for a time sufficient and underconditions suitable for target binding, which conditions can be selectedaccording to conditions under which target binding to the AB would beexpected. A negative selection can then be performed to identifycandidate AAs that are relatively decreased for target binding,including those which exhibit no detectably target binding. Thisselection can be accomplished by, for example, use of a detectablylabeled target, and subjecting the target-exposed population to flowcytometry analysis to sort into separate subpopulation those cells thatdisplay a candidate AA that exhibits no detectable target binding and/orwhich exhibit a relatively lower detectable signal. This subpopulationis thus enriched for cells having a candidate AA that exhibit decreasedor undetectable binding to target in the absence of cleavage.

(d) Detectable Labels

A detectable label and detectable moiety are used interchangeably torefer to a molecule capable of detection, including, but not limited to,radioactive isotopes, fluorescers, chemiluminescers, chromophores,enzymes, enzyme substrates, enzyme cofactors, enzyme inhibitors,chromophores, dyes, metal ions, metal sols, ligands (e.g., biotin,avidin, strepavidin or haptens) and the like. The term fluorescer refersto a substance or a portion thereof which is capable of exhibitingfluorescence in the detectable range. Exemplary detectable moietiessuitable for use as target labels include affinity tags and fluorescentproteins.

The term affinity tag is used herein to denote a peptide segment thatcan be attached to a target that can be detected using a molecule thatbinds the affinity tag and provides a detectable signal (e.g., afluorescent compound or protein). In principal, any peptide or proteinfor which an antibody or other specific binding agent is available canbe used as an affinity tag. Exemplary affinity tags suitable for useinclude, but are not limited to, a monocytic adaptor protein (MONA)binding peptide, a T7 binding peptide, a streptavidin binding peptide, apolyhistidine tract, protein A (Nilsson et al., EMBO J. 4:1075 (1985);Nilsson et al., Methods Enzymol. 198:3 (1991)), glutathione Stransferase (Smith and Johnson, Gene 67:31 (1988)), Glu-Glu affinity tag(Grussenmeyer et al., Proc. Natl. Acad. Sci. USA 82:7952 (1985)),substance P, FLAG peptide (Hopp et al., Biotechnology 6:1204 (1988)), orother antigenic epitope or binding domain. See, in general, Ford et al.,Protein Expression and Purification 2:95 (1991). DNA molecules encodingaffinity tags are available from commercial suppliers (e.g., PharmaciaBiotech, Piscataway, N.J.).

Any fluorescent polypeptide (also referred to herein as a fluorescentlabel) well known in the art is suitable for use as a detectable moietyor with an affinity tag of the peptide display scaffolds describedherein. A suitable fluorescent polypeptide will be one that can beexpressed in a desired host cell, such as a bacterial cell or amammalian cell, and will readily provide a detectable signal that can beassessed qualitatively (positive/negative) and quantitatively(comparative degree of fluorescence). Exemplary fluorescent polypeptidesinclude, but are not limited to, yellow fluorescent protein (YFP), cyanfluorescent protein (CFP), GFP, mRFP, RFP (tdimer2), HCRED, etc., or anymutant (e.g., fluorescent proteins modified to provide for enhancedfluorescence or a shifted emission spectrum), analog, or derivativethereof. Further suitable fluorescent polypeptides, as well as specificexamples of those listed herein, are provided in the art and are wellknown.

Biotin-based labels also find use in the methods disclosed herein.Biotinylation of target molecules and substrates is well known, forexample, a large number of biotinylation agents are known, includingamine-reactive and thiol-reactive agents, for the biotinylation ofproteins, nucleic acids, carbohydrates, carboxylic acids; see, e.g.,chapter 4, Molecular Probes Catalog, Haugland, 6th Ed. 1996, herebyincorporated by reference. A biotinylated substrate can be detected bybinding of a detectably labeled biotin binding partner, such as avidinor streptavidin. Similarly, a large number of haptenylation reagents arealso known.

(e) Screening Methods

Any suitable method that provides for separation and recovery of AAs ofinterest may be utilized. For example, a cell displaying an AA ofinterest may be separated by FACS, immunochromatography or, where thedetectable label is magnetic, by magnetic separation. As a result of theseparation, the population is enriched for cells that exhibit thedesired characteristic, e.g., exhibit binding to target followingcleavage or have decreased or no detectable binding to target in theabsence of cleavage.

For example, selection of candidate AAs having bound detectably labeledtarget can be accomplished using a variety of techniques known in theart. For example, flow cytometry (e.g., FACS®) methods can be used tosort detectably labeled candidate AAs from unlabeled candidate AAs. Flowcyomtery methods can be implemented to provide for more or lessstringent requirements in separation of the population of candidate AAs,e.g., by modification of gating to allow for dimmer or to requirebrighter cell populations in order to be separated into the secondpopulation for further screening.

In another example, immunoaffinity chromatography can be used toseparate target-bound candidate AAs from those that do not bind target.For example, a support (e.g., column, magnetic beads) having boundanti-target antibody can be contacted with the candidate AAs that havebeen exposed to protease and to target. Candidate AAs having boundtarget bind to the anti-target antibody, thus facilitating separationfrom candidate AAs lacking bound target. Where the screening step is toprovide for a population enriched for uncleaved candidate AAs that haverelatively decreased target binding or no detectable target binding(e.g., relative to other candidate AAs), the subpopulation of interestis those members that lack or have a relatively decreased detectablysignal for bound target. For example, where an immunoaffinity techniqueis used in such negative selection for bound target, the subpopulationof interest is that which is not bound by the anti-target support.

(f) Screening for Dual Target-Binding AAs

Methods for screening disclosed herein can be readily adapted toidentify dual target-binding AAs having two ABs. In general, the methodinvolves a library containing a plurality of candidate AAs, wherein eachmember of said plurality comprises a first AB, a second AB, a first CMand/or a second CM, a first MM, and/or a second MM. The library iscontacted with target capable of binding at least the first AB and acleaving agent capable of cleaving the first CM. A first population ofmembers of the library is selected for binding the target in thepresence of the cleaving agent (e.g., protease for the CM). Thisselected population is then subjected to the negative screen above, inwhich binding of target to the library members in the absence of thecleaving agent is assessed. A second population of members is thengenerated by depleting the subpopulation of members that bind to saidtarget in the absence of the cleaving agent. This can be accomplishedby, for example, sorting members that are not bound to target away fromthose that are bound to target, as determined by detection of adetectably labeled target. This method thus provides for selection ofcandidate AAs which exhibit decreased binding to the target in theabsence of the cleaving agent as compared to binding to said target inthe presence of the cleaving agent. This method can be repeated for bothtargets.

Exemplary Variations of the Screening Methods to Select for CandidateAAs

The above methods may be modified to select for populations and librarymembers that demonstrate desired characteristics.

(a) Determination of the Masking Efficiency of MMs

Masking efficiency of MMs is determined by at least two parameters:affinity of the MM for antibody or fragment thereof and the spatialrelationship of the MM relative to the binding interface of the AB toits target.

Regarding affinity, by way of example, an MM may have high affinity butonly partially inhibit the binding site on the AB, while another MM mayhave a lower affinity for the AB but fully inhibit target binding. Forshort time periods, the lower affinity MM may show sufficient masking;in contrast, over time, that same MM may be displaced by the target (dueto insufficient affinity for the AB).

In a similar fashion, two MMs with the same affinity may show differentextents of masking based on how well they promote inhibition of thebinding site on the AB or prevention of the AB from binding its target.In another example, a MM with high affinity may bind and change thestructure of the AB so that binding to its target is completelyinhibited while another MM with high affinity may only partially inhibitbinding. As a consequence, discovery of an effective MM cannot be basedonly on affinity but can include an empirical measure of maskingefficiency. The time-dependent target displacement of the MM in the AAcan be measured to optimize and select for MMs. A novel TargetDisplacement Assay (TDA) is described herein for this purpose.

The TDA assay can be used for the discovery and validation ofefficiently masked AAs comprises empirical determination of maskingefficiency, comparing the ability of the masked AB to bind the target inthe presence of target to the ability of the unmasked and/or parental ABto bind the target in the presence of the target. The binding efficiencycan be expressed as a % of equilibrium binding, as compared tounmasked/parental AB binding. When the AB is modified with a MM and isin the presence of the target, specific binding of the AB to its targetcan be reduced or inhibited, as compared to the specific binding of theAB not modified with an MM or the parental AB to the target. Whencompared to the binding of the AB not modified with an MM or theparental AB to the target, the AB's ability to bind the target whenmodified with an MM can be reduced by at least 50%, 60%, 70%, 80%, 90%,92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% and even 100% for at least 2, 4,6, 8, 12, 28, 24, 30, 36, 48, 60, 72, 84, 96, hours, or 5, 10, 15, 30,45, 60, 90, 120, 150, 180 days, or 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12months or greater when measured in vivo or in a Target Displacement invitro immunosorbant assay, as described herein.

(b) Iterative Screens to Identify and/or Optimize AA Elements

The methods and candidate AA libraries described herein can be readilyadapted to provide for identification and/or optimization of one or moreelements of an AA. For example, candidate AAs that vary with respect toany one or more of AB, CM, linkers, and the like can be produced andsubjected to the screening methods described herein.

(c) Reducing Agent-Activatable AAs

While the methods above describe screening methods for identifying AAs,it should be understood that an AA or candidate AA with a CM that canfacilitate formation of a cysteine-cysteine disulfide bond in an AA andcan also be subjected to the screening methods disclosed herein. SuchAAs may or may not further include a CM (which may be the same ordifferent CM) that may or may not comprise a protease substrate. Inthese embodiments, the positive screen described above may be conductedby exposing an AA or candidate AA to a reducing agent (e.g., to reducingconditions) capable of cleaving the disulfide bond of thecysteine-cysteine pair of the AA. The negative screen can then beconducted in the absence of the reducing conditions. As such, a libraryproduced having may be enriched for AAs which are activatable byexposure to disulfide bond reducing conditions.

(d) Photo-Activatable AAs

While the methods above describe screening methods for identifying AAs,it should be understood that an AA or candidate AA with a CM that isphoto-sensitive, and can be activated upon photolysis are also provided.In these embodiments, the positive screen described above may beconducted by exposing an AA or candidate AA to light. The negativescreen can then be conducted in the absence of light. As such, a libraryproduced having may be enriched for AAs which are activatable byexposure to light.

(e) Number of Cycles and Scaffold Free Screening of AAs

By increasing the number of cycles of the above methods, populations andlibrary members that demonstrate improved switching characteristics canbe identified. Any number of cycles of screening can be performed.

In addition, individual clones of candidate AAs can be isolated andsubjected to screening so as to determine the dynamic range of thecandidate AA. Candidate AAs can also be tested for a desired switchablephenotype separate from the scaffold, i.e., the candidate AA can beexpressed or otherwise generated separate from the display scaffold, andthe switchable phenotype of the candidate AA assessed in the absence ofthe scaffold and, where desired, in a cell-free system (e.g., usingsolubilized AA).

(f) Optimization of AA Components and Switching Activity

The above methods may be modified to optimize the performance of an AA,e.g., an AA identified in the screening method described herein. Forexample, where it is desirable to optimize the performance of themasking moiety, e.g., to provide for improved inhibition of targetbinding of the AB in the uncleaved state, the amino acid sequences ofthe AB and the CM may be fixed in a candidate AA library, and the MMvaried such that members of a library have variable MMs relative to eachother. The MM may be optimized in a variety of ways including alterationin the number and or type of amino acids that make up the MM. Forexample, each member of the plurality of candidate AAs may comprise acandidate MM, wherein the candidate MM comprises at least one cysteineamino acid residue and the remaining amino acid residues are variablebetween the members of the plurality. In a further example, each memberof the plurality of candidate AAs may comprise a candidate MM, whereinthe candidate MM comprises a cysteine amino acid residue and a randomsequence of amino acid residues, e.g., a random sequence of 5 aminoacids.

(g) Selection for Expanded Dynamic Range

As noted above, AAs having a desired dynamic range with respect totarget binding in the unmasked/cleaved versus masked/uncleaved state arealso of interest. Such AAs are those that, for example, have nodetectable binding in the presence of target at physiological levelsfound at treatment and non-treatment sites in a subject but which, oncecleaved by protease, exhibit high affinity and/or high avidity bindingto target. The greater the dynamic range of an AA, the better theswitchable phenotype of the AA. Thus AAs can be optimized to select forthose having an expanded dynamic range for target binding in thepresence and absence of a cleaving agent.

The screening methods described herein can be modified so as to enhanceselection of AAs having a desired and/or optimized dynamic range. Ingeneral, this can be accomplished by altering the concentrations oftarget utilized in the positive selection and negative selection stepsof the method such that screening for target binding of AAs exposed toprotease (i.e., the screening population that includes cleaved AAs) isperformed using a relatively lower target concentration than whenscreening for target binding of uncleaved AAs. Accordingly, the targetconcentration is varied between the steps so as to provide a selectivepressure toward a switchable phenotype. Where desired, the difference intarget concentrations used at the positive and negative selection stepscan be increased with increasing cycle number.

Use of a relatively lower concentration of target in the positiveselection step can serve to drive selection of those AA members thathave improved target binding when in the cleaved state. For example, thescreen involving protease-exposed AAs can be performed at a targetconcentration that is from about 2 to about 100 fold lower, about 2 to50 fold lower, about 2 to 20 fold lower, about 2 to 10-fold lower, orabout 2 to 5-folder lower than the K_(d) of the AB-target interaction.As a result, after selection of the population for target-bound AAs, theselected population will be enriched for AAs that exhibit higheraffinity and/or avidity binding relative to other AAs in the population.

Use of a relatively higher concentration of target in the negativeselection step can serve to drive selection of those AA members thathave decreased or no detectable target binding when in the uncleavedstate. For example, the screen involving AAs that have not been exposedto protease (in the negative selection step) can be performed at atarget concentration that is from about 2 to about 100 fold higher,about 2 to 50 fold higher, about 2 to 20 fold higher, about 2 to 10-foldhigher, or about 2 to 5-folder higher, than the K_(d) of the AB-targetinteraction. As a result, after selection of the population for AAs thatdo not detectably bind target, the selected population will be enrichedfor AAs that exhibit lower binding for target when in the uncleavedstate relative to other uncleaved AAs in the population. Stateddifferently, after selection of the population for AAs that do notdetectably bind target, the selected population will be enriched for AAsfor which target binding to AB is inhibited, e.g., due to masking of theAB from target binding.

Where the AA is a dual target-binding AA, the screening method describedabove can be adapted to provide for AAs having a desired dynamic rangefor a first target that is capable of binding a first AB and for asecond target that is capable of binding a second AB. Target binding toan AB that is located on a portion of the AA that is cleaved away fromthe AA presented on a display scaffold can be evaluated by assessingformation of target-AB complexes in solution (e.g., in the culturemedium), e.g., immunochromatography having an anti-AA fragment antibodyto capture cleaved fragment, then detecting bound, detectably labeledtarget captured on the column.

(h) Testing of Soluble AAs

Candidate AAs can be tested for their ability to maintain a switchablephenotype while in soluble form. One such method involves theimmobilization of target to support (e.g., an array, e.g., a Biacore™CM5 sensor chip surface). Immobilization of a target of interest can beaccomplished using any suitable techniques (e.g., standard aminecoupling). The surface of the support can be blocked to reducenon-specific binding. Optionally, the method can involve use of acontrol (e.g., a support that does not contain immobilized target (e.g.,to assess background binding to the support) and/or contains a compoundthat serves as a negative control (e.g., to assess specificity ofbinding of the candidate AA to target versus non-target).

After the target is covalently immobilized, the candidate AA iscontacted with the support under conditions suitable to allow forspecific binding to immobilized target. The candidate AA can becontacted with the support-immobilized target in the presence and in theabsence of a suitable cleavage agent in order to assess the switchablephenotype. Assessment of binding of the candidate AA in the presence ofcleavage agent as compared to in the absence of cleavage agent and,optionally, compared to binding in a negative control provides a bindingresponse, which in turn is indicative of the switchable phenotype.

(i) Screening for Individual Moieties for Use in Candidate AAs

It may be desirable to screen separately for one or more of the moietiesof a candidate AA, e.g., an AB, MM or CM, prior to testing the candidateAA for a switchable phenotype. For example, known methods of identifyingpeptide substrates cleavable by specific proteases can be utilized toidentify CMs for use in AAs designed for activation by such proteases.In addition a variety of methods are available for identifying peptidesequences which bind to a target of interest. These methods can be used,for example, to identify ABs which binds to a particular target or toidentify a MM which binds to a particular AB.

The above methods include, for example, methods in which a moiety of acandidate AA, e.g., an AB, MM or CM, is displayed using a replicablebiological entity.

(j) Automated Screening Methods

In certain embodiments the screening methods described herein areautomated to provide convenient, real time, high volume methods ofscreening a library of AAs for a desired switchable activity. Automatedmethods can be designed to provide for iterative rounds of positive andnegative selection, with the selected populations being separated andautomatically subjected to the next screen for a desired number ofcycles.

Assessing candidate AAs in a population may be carried out over timeiteratively, following completion of a positive selection step, anegative selection step, or both. In addition, information regarding theaverage dynamic range of a population of candidate AAs at selectedtarget concentrations in the positive and negative selection steps canbe monitored and stored for later analysis, e.g. so as to assess theeffect of selective pressure of the different target concentrations.

In some embodiments, a executable platform such as a computer softwareproduct can control operation of the detection and/or measuring meansand can perform numerical operations relating to the above-describedsteps, and generate a desired output (e.g., flow cytometry analysis,etc.). Computer program product comprises a computer readable storagemedium having computer-readable program code means embodied in themedium. Hardware suitable for use in such automated apparatus will beapparent to those of skill in the art, and may include computercontrollers, automated sample handlers, fluorescence measurement tools,printers and optical displays. The measurement tool may contain one ormore photodetectors for measuring the fluorescence signals from sampleswhere fluorescently detectable molecules are utilized. The measurementtool may also contain a computer-controlled stepper motor so that eachcontrol and/or test sample can be arranged as an array of samples andautomatically and repeatedly positioned opposite a photodetector duringthe step of measuring fluorescence intensity.

The measurement tool (e.g., FACS) can be operatively coupled to ageneral purpose or application-specific computer controller. Thecontroller can comprise a computer program produce for controllingoperation of the measurement tool and performing numerical operationsrelating to the above-described steps. The controller may accept set-upand other related data via a file, disk input or data bus. A display andprinter may also be provided to visually display the operationsperformed by the controller. It will be understood by those having skillin the art that the functions performed by the controller may berealized in whole or in part as software modules running on a generalpurpose computer system. Alternatively, a dedicated stand-alone systemwith application specific integrated circuits for performing the abovedescribed functions and operations may be provided.

Methods of Use of AAs in Therapy

AAs can be incorporated into pharmaceutical compositions containing, forexample, a therapeutically effective amount of an AA of interest and acarrier that is a pharmaceutically acceptable excipient (also referredto as a pharmaceutically acceptable carrier). Many pharmaceuticallyacceptable excipients are known in the art, are generally selectedaccording to the route of administration, the condition to be treated,and other such variables that are well understood in the art.Pharmaceutically acceptable excipients have been amply described in avariety of publications, including, for example, A. Gennaro (2000)Remington: The Science and Practice of Pharmacy, 20th edition,Lippincott, Williams, & Wilkins; Pharmaceutical Dosage Forms and DrugDelivery Systems (1999) H. C. Ansel et al., eds., 7^(th) ed.,Lippincott, Williams, & Wilkins; and Handbook of PharmaceuticalExcipients (2000) A. H. Kibbe et al., eds., 3^(rd) ed. Amer.Pharmaceutical Assoc. Pharmaceutical compositions can also include othercomponents such as pH adjusting and buffering agents, tonicity adjustingagents, stabilizers, wetting agents and the like. In some embodiments,nanoparticles or liposomes carry a pharmaceutical composition comprisingan AA.

Suitable components for pharmaceutical compositions of AAs can be guidedby pharmaceutical compositions that may be already available for an ABof the AA. For example, where the AA includes an antibody to EGFR,TNFalpha, CD11a, CSFR, CTLA-4, EpCAM, VEGF, CD40, CD20, Notch 1, Notch2, Notch 3, Notch 4, Jagged 1, Jagged 2, CD52, MUC1, IGF1R, transferrin,gp130, VCAM-1, CD44, DLL4, or IL4, for example, such AAs can beformulated in a pharmaceutical formulation according to methods andcompositions suitable for use with that antibody.

In general, pharmaceutical formulations of one or more AAs are preparedfor storage by mixing the AA having a desired degree of purity withoptional physiologically acceptable carriers, excipients or stabilizers(Remington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)),in the form of lyophilized formulations or aqueous solutions. Acceptablecarriers, excipients, or stabilizers are nontoxic to recipients at thedosages and concentrations employed, and include buffers such asphosphate, citrate, and other organic acids; antioxidants includingascorbic acid and methionine; preservatives (such asoctadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptide; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN™, PLURONICS™ or polyethylene glycol (PEG).

The formulations to be used for in vivo administration must be sterile.This is readily accomplished by filtration through sterile filtrationmembranes. Pharmaceutical formulations may also contain more than oneactive compound as necessary for the particular indication beingtreated, where the additional active compounds generally are those withactivities complementary to an AA. Such compounds are suitably presentin combination in amounts that are effective for the purpose intended.

The pharmaceutical formulation can be provided in a variety of dosageforms such as a systemically or local injectable preparation. Thecomponents can be provided in a carrier such as a microcapsule, e.g.,such as that prepared by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

Sustained-release preparations are also within the scope of anAA-containing formulations. Exemplary sustained-release preparations caninclude semi-permeable matrices of solid hydrophobic polymers containingthe AA, which matrices are in the form of shaped articles, e.g., films,or microcapsule. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and γ-ethyl-L-glutamate, non-degradableethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymerssuch as the LUPRON DEPOT™ (inj ectable microspheres composed of lacticacid-glycolic acid copolymer and leuprolide acetate), andpoly-D-(−)-3-hydroxybutyric acid. While polymers such as ethylene-vinylacetate and lactic acid-glycolic acid enable release of molecules forover 100 days, certain hydrogels release proteins for shorter timeperiods.

When encapsulated AAs remain in the body for a long time, they maydenature or aggregate as a result of exposure to moisture atphysiological temperature (−37° C.), resulting in decreased biologicalactivity and possible changes in immunogenicity. Rational strategies canbe devised for stabilization depending on the mechanism involved. Forexample, if the aggregation mechanism is discovered to be undesirableintermolecular S—S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

AAs can be conjugated to delivery vehicles for targeted delivery of anactive agent that serves a therapeutic purpose. For example, AAs can beconjugated to nanoparticles or liposomes having drugs encapsulatedtherein or associated therewith. In this manner, specific, targeteddelivery of the drug can be achieved. Methods of linking polypeptides toliposomes are well known in the art and such methods can be applied tolink AAs to liposomes for targeted and or selective delivery of liposomecontents. By way of example, polypeptides can be covalently linked toliposomes through thioether bonds. PEGylated gelatin nanoparticles andPEGylated liposomes have also been used as a support for the attachmentof polypeptides, e.g., single chain antibodies. See, e.g., Immordino etal. (2006) Int J Nanomedicine. September; 1(3): 297-315, incorporated byreference herein for its disclosure of methods of conjugatingpolypeptides, e.g., antibody fragments, to liposomes.

(a) Methods of Treatment

AAs described herein can be selected for use in methods of treatment ofsuitable subjects according to the CM-AB combination provided in the AA.The AA can be administered by any suitable means, including oral,parenteral, subcutaneous, intraperitoneal, intrapulmonary, andintranasal, and, if desired for local injection (e.g., at the site of asolid tumor). Parenteral administration routes include intramuscular,intravenous, intraarterial, intraperitoneal, or subcutaneousadministration.

The term treatment site or disease site is meant to refer to a site atwhich an AA is designed to be switchable, as described herein, e.g., asite at which a target for one or both ABs of an AA and a cleaving agentcapable of cleaving a CM of the AA are co-localized, as pictoriallyrepresented in FIG. 2. Treatment sites include tissues that can beaccessed by local administration (e.g., injection, infusion (e.g., bycatheter), etc.) or by systemic administration (e.g., administration toa site remote from a treatment site). Treatment sites include those thatare relatively biologically confined (e.g., an organ, sac, tumor site,and the like).

The appropriate dosage of an AA will depend on the type of disease to betreated, the severity and course of the disease, the patient's clinicalhistory and response to the AA, and the discretion of the attendingphysician. AAs can suitably be administered to the patient at one timeor over a series of treatments. AAs can be administered along with othertreatments and modes of therapies, other pharmaceutical agents, and thelike.

Depending on the type and severity of the disease, about 1 μg/kg to 15mg/kg (e.g., 0.1-20 mg/kg) of an AA can serve as an initial candidatedosage for administration to the patient, whether, for example, by oneor more separate administrations, or by continuous infusion. A typicaldaily dosage might range from about 1 μg/kg to 100 mg/kg or more,depending on factors such as those mentioned herein. For repeatedadministrations over several days or longer, depending on the condition,the treatment is sustained until a desired suppression of diseasesymptoms occurs. However, other dosage regimens may be useful.

The AA composition will be formulated, dosed, and administered in afashion consistent with good medical practice. Factors for considerationin this context include the particular disorder being treated, theparticular mammal being treated, the clinical condition of theindividual patient, the cause of the disorder, the site of delivery ofthe AA, the method of administration, the scheduling of administration,and other factors known to medical practitioners. The therapeuticallyeffective amount of an AA to be administered will be governed by suchconsiderations, and is the minimum amount necessary to prevent,ameliorate, or treat a disease or disorder.

Generally, alleviation or treatment of a disease or disorder involvesthe lessening of one or more symptoms or medical problems associatedwith the disease or disorder. For example, in the case of cancer, thetherapeutically effective amount of the drug can accomplish one or acombination of the following: reduce the number of cancer cells; reducethe tumor size; inhibit (i.e., to decrease to some extent and/or stop)cancer cell infiltration into peripheral organs; inhibit tumormetastasis; inhibit, to some extent, tumor growth; and/or relieve tosome extent one or more of the symptoms associated with the cancer. Insome embodiments, a composition of this invention can be used to preventthe onset or reoccurrence of the disease or disorder in a subject ormammal.

AAs can be used in combination (e.g., in the same formulation or inseparate formulations) with one or more additional therapeutic agents ortreatment methods (combination therapy). An AA can be administered inadmixture with another therapeutic agent or can be administered in aseparate formulation. Therapeutic agents and/or treatment methods thatcan be administered in combination with an AA, and which are selectedaccording to the condition to be treated, include surgery (e.g.,surgical removal of cancerous tissue), radiation therapy, bone marrowtransplantation, chemotherapeutic treatment, certain combinations of theforegoing, and the like.

(b) Use of AAs in Diseased Tissue Versus Healthy Tissue

The AAs of the present invention, when localized to a healthy tissue,show little or no activation and the AB remains in a ‘masked’ state, orotherwise exhibits little or no binding to the target. However, in adiseased tissue, in the presence of a disease-specific protease, forexample, capable of cleaving the CM of the AA, the AB becomes ‘unmasked’or can specifically bind the target.

A healthy tissue refers to a tissue that produces little or nodisease-specific agent capable of specifically cleaving or otherwisemodifying the CM of the AA, for example a disease-specific protease, adisease-specific enzyme, or a disease-specific reducing agent. Adiseased tissue refers to a tissue that produces a disease-specificagent capable of specifically cleaving or otherwise modifying the CM ofthe AA, for example a disease-specific protease, a disease-specificenzyme, or a disease-specific reducing agent.

(c) Use of AAs in Diseased Tissue at Different Stages of a Disease

In some embodiments, the AAs described herein are coupled to more thanone CM. Such an AA can be activated in different stages of a disease, oractivated in different compartments of the diseased tissue. By way ofexample, an AB coupled to both a MMP-9 cleavable CM and a cathepsinD-cleavable CM can be activated in an early stage tumor and in a latestage, necrosing tumor. In the early stage tumor, the CM can be cleavedand the AA unmasked by MMP-9. In the late stage tumor, the CM can becleaved and the AA unmasked by cathepsin D which is upregulated in thedying center of late stage tumors. In another exemplary embodiment an ABcoupled to an MM and to a MMP-9-activatable CM and a caspase-activatableCM can be cleaved at both early and late stage tumors. In anotherplasmin at active sites of angiogenesis (early stage tumor) can cleave aplasmin-cleavable CM and legumain in disease tissues with invadingmacrophages can cleave a leugamain-specific CM in a late stage tumor.

(d) Use of AAs in Anti-Angiogenic Therapies

In an exemplary embodiment where the AA contains an AB that binds amediator of angiogenesis such as EGFR, TNFalpha, CD11a, CSFR, CTLA-4,EpCAM, VEGF, CD40, CD20, Notch 1, Notch 2, Notch 3, Notch 4, Jagged 1,Jagged 2, CD52, MUC1, IGF1R, transferrin, gp130, VCAM-1, CD44, DLL4, orIL4, the AA finds use in treatment of conditions in which inhibition ofangiogenesis is desired, particularly those conditions in whichinhibition of VEGF is of interest. VEGF-binding AAs can include dualtarget binding AAs having an AB that binds to VEGF as well as an AB thatbinds to a second growth factor, such as a fibroblast growth factor(e.g., FGF-2), and inhibits FGF activity. Such dual target binding AAsthus can be designed to provide for inhibition of twoangiogenesis-promoting factors, and which are activatable by a cleavingagent (e.g., enzyme, such as a MMP or other enzymes such as onepresented in Table 3) which co-localizes at a site of aberrantangiogenesis.

Angiogenesis-inhibiting AAs find use in treatment of solid tumors in asubject (e.g., human), particularly those solid tumors that have anassociated vascular bed that feeds the tumor such that inhibition ofangiogenesis can provide for inhibition or tumor growth. Anti-VEGF-basedanti-angiogenesis AAs also find use in other conditions having one ormore symptoms amenable to therapy by inhibition of abnormalangiogenesis.

In general, abnormal angiogenesis occurs when new blood vessels eithergrow excessively, insufficiently or inappropriately (e.g., the location,timing or onset of the angiogenesis being undesired from a medicalstandpoint) in a diseased state or such that it causes a diseased state.Excessive, inappropriate or uncontrolled angiogenesis occurs when thereis new blood vessel growth that contributes to the worsening of thediseased state or causes a diseased state, such as in cancer, especiallyvascularized solid tumors and metastatic tumors (including colon, lungcancer (especially small-cell lung cancer), or prostate cancer),diseases caused by ocular neovascularisation, especially diabeticblindness, retinopathies, primarily diabetic retinopathy or age-inducedmacular degeneration and rubeosis; psoriasis, psoriatic arthritis,haemangioblastoma such as haemangioma; inflammatory renal diseases, suchas glomerulonephritis, especially mesangioproliferativeglomerulonephritis, haemolytic uremic syndrome, diabetic nephropathy orhypertensive neplirosclerosis; various imflammatory diseases, such asarthritis, especially rheumatoid arthritis, inflammatory bowel disease,psorsasis, sarcoidosis, arterial arteriosclerosis and diseases occurringafter transplants, endometriosis or chronic asthma and other conditionsthat will be readily recognized by the ordinarily skilled artisan. Thenew blood vessels can feed the diseased tissues, destroy normal tissues,and in the case of cancer, the new vessels can allow tumor cells toescape into the circulation and lodge in other organs (tumormetastases).

AA-based anti-angiogenesis therapies can also find use in treatment ofgraft rejection, lung inflammation, nephrotic syndrome, preeclampsia,pericardial effusion, such as that associated with pericarditis, andpleural effusion, diseases and disorders characterized by undesirablevascular permeability, e.g., edema associated with brain tumors, ascitesassociated with malignancies, Meigs' syndrome, lung inflammation,nephrotic syndrome, pericardial effusion, pleural effusion, permeabilityassociated with cardiovascular diseases such as the condition followingmyocardial infarctions and strokes and the like.

Other angiogenesis-dependent diseases that may be treated usinganti-angiogenic AAs as described herein include angiofibroma (abnormalblood of vessels which are prone to bleeding), neovascular glaucoma(growth of blood vessels in the eye), arteriovenous malformations(abnormal communication between arteries and veins), nonunion fractures(fractures that will not heal), atherosclerotic plaques (hardening ofthe arteries), pyogenic granuloma (common skin lesion composed of bloodvessels), scleroderma (a form of connective tissue disease), hemangioma(tumor composed of blood vessels), trachoma (leading cause of blindnessin the third world), hemophilic joints, vascular adhesions andhypertrophic scars (abnormal scar formation).

Amounts of an AA for administration to provide a desired therapeuticeffect will vary according to a number of factors such as thosediscussed above. In general, in the context of cancer therapy, atherapeutically effective amount of an AA is an amount that that iseffective to inhibit angiogenesis, and thereby facilitate reduction of,for example, tumor load, atherosclerosis, in a subject by at least about5%, at least about 10%, at least about 20%, at least about 25%, at leastabout 50%, at least about 75%, at least about 85%, or at least about90%, up to total eradication of the tumor, when compared to a suitablecontrol. In an experimental animal system, a suitable control may be agenetically identical animal not treated with the agent. Innon-experimental systems, a suitable control may be the tumor loadpresent before administering the agent. Other suitable controls may be aplacebo control.

Whether a tumor load has been decreased can be determined using anyknown method, including, but not limited to, measuring solid tumor mass;counting the number of tumor cells using cytological assays;fluorescence-activated cell sorting (e.g., using antibody specific for atumor-associated antigen) to determine the number of cells bearing agiven tumor antigen; computed tomography scanning, magnetic resonanceimaging, and/or x-ray imaging of the tumor to estimate and/or monitortumor size; measuring the amount of tumor-associated antigen in abiological sample, e.g., blood or serum; and the like.

In some embodiments, the methods are effective to reduce the growth rateof a tumor by at least about 5%, at least about 10%, at least about 20%,at least about 25%, at least about 50%, at least about 75%, at leastabout 85%, or at least about 90%, up to total inhibition of growth ofthe tumor, when compared to a suitable control. Thus, in theseembodiments, effective amounts of an AA are amounts that are sufficientto reduce tumor growth rate by at least about 5%, at least about 10%, atleast about 20%, at least about 25%, at least about 50%, at least about75%, at least about 85%, or at least about 90%, up to total inhibitionof tumor growth, when compared to a suitable control. In an experimentalanimal system, a suitable control may be tumor growth rate in agenetically identical animal not treated with the agent. Innon-experimental systems, a suitable control may be the tumor load ortumor growth rate present before administering the agent. Other suitablecontrols may be a placebo control.

Whether growth of a tumor is inhibited can be determined using any knownmethod, including, but not limited to, an in vivo assay for tumorgrowth; an in vitro proliferation assay; a ³H-thymidine uptake assay;and the like.

(e) Use of AAs in Anti-Inflammatory Therapies

In another exemplary embodiment where the AA contains an AB that bindsmediators of inflammation such as interleukins, the AA finds use intreatment of related conditions. Interleukin-binding AAs can includedual target binding AAs having an AB that binds to for example IL12 aswell as an AB that binds to IL23, or an AA where a first AB binds toIL17 and a second AB binds to IL23. Such dual target binding AAs thuscan be designed to provide for mediation of inflammation, and which areactivatable by a cleaving agent (e.g., enzyme, such as a MMP or otherenzyme such as one presented in Table 3) which co-localizes at a site ofinflammation.

Non-Therapeutic Methods of Using AAs

AAs can also be used in diagnostic and/or imaging methods. For example,AAs having an enzymatically cleavable CM can be used to detect thepresence or absence of an enzyme that is capable of cleaving the CM.Such AAs can be used in diagnostics, which can include in vivo detection(e.g., qualitative or quantitative) of enzyme activity (or, in someembodiments, an environment of increased reduction potential such asthat which can provide for reduction of a disulfide bond) accompanied bypresence of a target of interest through measured accumulation ofactivated AAs in a given tissue of a given host organism.

For example, the CM can be selected to be a protease substrate for aprotease found at the site of a tumor, at the site of a viral orbacterial infection at a biologically confined site (e.g., such as in anabscess, in an organ, and the like), and the like. The AB can be onethat binds a target antigen. Using methods familiar to one skilled inthe art, a detectable label (e.g., a fluorescent label) can beconjugated to an AB or other region of an AA. Suitable detectable labelsare discussed in the context of the above screening methods andadditional specific examples are provided below. Using an AB specific toa protein or peptide of the disease state, along with a protease whoseactivity is elevated in the disease tissue of interest, AAs will exhibitincreased rate of binding to disease tissue relative to tissues wherethe CM specific enzyme is not present at a detectable level or ispresent at a lower level than in disease tissue. Since small proteinsand peptides are rapidly cleared from the blood by the renal filtrationsystem, and because the enzyme specific for the CM is not present at adetectable level (or is present at lower levels in non-diseasedtissues), accumulation of activated AA in the diseased tissue isenhanced relative to non-disease tissues.

In another example, AAs can be used in to detect the presence or absenceof a cleaving agent in a sample. For example, where the AA contains a CMsusceptible to cleavage by an enzyme, the AA can be used to detect(either qualitatively or quantitatively) the presence of an enzyme inthe sample. In another example, where the AA contains a CM susceptibleto cleavage by reducing agent, the AA can be used to detect (eitherqualitatively or quantitatively) the presence of reducing conditions ina sample. To facilitate analysis in these methods, the AA can bedetectably labeled, and can be bound to a support (e.g., a solidsupport, such as a slide or bead). The detectable label can bepositioned on a portion of the AA that is released following cleavage.The assay can be conducted by, for example, contacting the immobilized,detectably labeled AA with a sample suspected of containing an enzymeand/or reducing agent for a time sufficient for cleavage to occur, thenwashing to remove excess sample and contaminants. The presence orabsence of the cleaving agent (e.g., enzyme or reducing agent) in thesample is then assessed by a change in detectable signal of the AA priorto contacting with the sample (e.g., a reduction in detectable signaldue to cleavage of the AA by the cleaving agent in the sample and theremoval of an AA fragment to which the detectable label is attached as aresult of such cleavage.

Such detection methods can be adapted to also provide for detection ofthe presence or absence of a target that is capable of binding the AB ofthe AA. Thus, the in vitro assays can be adapted to assess the presenceor absence of a cleaving agent and the presence or absence of a targetof interest. The presence or absence of the cleaving agent can bedetected by a decrease in detectable label of the AA as described above,and the presence or absence of the target can be detected by detectionof a target-AB complex, e.g., by use of a detectably labeled anti-targetantibody.

As discussed above, the AAs disclosed herein can comprise a detectablelabel. In one embodiment, the AA comprises a detectable label which canbe used as a diagnostic agent. Non-limiting examples of detectablelabels that can be used as diagnostic agents include imaging agentscontaining radioisotopes such as indium or technetium; contrastingagents for MRI and other applications containing iodine, gadolinium oriron oxide; enzymes such as horse radish peroxidase, alkalinephosphatase, or β-galactosidase; fluorescent substances and fluorophoressuch as GFP, europium derivatives; luminescent substances such asN-methylacrydium derivatives or the like.

The rupture of vulnerable plaque and the subsequent formation of a bloodclot are believed to cause the vast majority of heart attacks. Effectivetargeting of vulnerable plaques can enable the delivery of stabilizingtherapeutics to reduce the likelihood of rupture.

VCAM-1 is upregulated both in regions prone to atherosclerosis as wellas at the borders of established lesions. Iiyama, et al. (1999)Circulation Research, μm Heart Assoc. 85: 199-207. Collagenases, such asMMP-1, MMP-8 and MMP-13, are overexpressed in human atheroma which maycontribute to the rupture of atheromatous plaques. Fricker, J. (2002)Drug Discov Today 7(2): 86-88.

In one example, AAs disclosed herein find use in diagnostic and/orimaging methods designed to detect and/or label atherosclerotic plaques,e.g., vulnerable atherosclerotic plaques. By targeting proteinsassociated with atherosclerotic plaques, AAs can be used to detectand/or label such plaques. For example, AAs comprising an anti-VCAM-1 ABand a detectable label find use in methods designed to detect and/orlabel atherosclerotic plaques. These AAs can be tested in animal models,such as ApoE mice.

Biodistribution Considerations

The therapeutic potential of the compositions described herein allow forgreater biodistribution and bioavailability of the modified AB or theAA. The compositions described herein provide an antibody therapeutichaving an improved bioavailability wherein the affinity of binding ofthe antibody therapeutic to the target is lower in a healthy tissue whencompared to a diseased tissue. A pharmaceutical composition comprisingan AB coupled to a MM can display greater affinity to the target in adiseased tissue than in a healthy tissue. In preferred embodiments, theaffinity in the diseased tissue is 5-10,000,000 times greater than theaffinity in the healthy tissue.

Generally stated, the present disclosure provides for an antibodytherapeutic having an improved bioavailability wherein the affinity ofbinding of the antibody therapeutic to its target is lower in a firsttissue when compared to the binding of the antibody therapeutic to itstarget in a second tissue. By way of example in various embodiments, thefirst tissue is a healthy tissue and the second tissue is a diseasedtissue; or the first tissue is an early stage tumor and the secondtissue is a late stage tumor; the first tissue is a benign tumor and thesecond tissue is a malignant tumor; the first tissue and second tissueare spatially separated; or in a specific example, the first tissue isepithelial tissue and the second tissue is breast, head, neck, lung,pancreatic, nervous system, liver, prostate, urogenital, or cervicaltissue.

EXAMPLES Example 1: Screening of Candidate Masking Moieties (MMs)

In order to produce compositions comprising antibodies and fragmentsthereof (AB) coupled to MMs with desired optimal binding anddissociation characteristics, libraries of candidate MMs are screened.MMs having different variable amino acid sequences, varying positions ofthe cysteine, various lengths, and the like are generated. Candidate MMsare tested for their affinity of binding to ABs of interest. Preferably,MMs not containing the native amino acid sequence of the binding partnerof the AB are selected for construction of the modified antibodies.

Affinity maturation of MMs for ABs of interest to select for MMs with anaffinity of about 1-10 nM is carried out.

Example 2: Screening of Modified Antibody and Activatable Antibody (AA)Libraries

In order to identify modified antibodies and AAs having desiredswitching characteristics (i.e., decreased target binding when in anmasked and/or uncleaved conformation relative to target binding when ina masked and/or cleaved conformation), libraries of candidate modifiedantibodies and candidate AAs having different variable amino acidsequences in the masking moieties (MMs), varying positions of thecysteine in the MM, various linker lengths, and various points ofattachment to the parent AB are generated.

A scheme for screening/sorting method to identify candidate AAs thatdisplay the switchable phenotype is provided here. The libraries areintroduced via expression vectors resulting in display of the AAs on thesurface of bacterial cells. After expansion of the libraries by culture,cells displaying the AA polypeptides are then treated with theappropriate enzyme or reducing agent to provide for cleavage orreduction of the CM. Treated cells are then contacted with fluorescentlylabeled target and the cells are sorted by FACS to isolate cellsdisplaying AAs which bind target after cleavage/reduction. The cellsthat display target-binding constructs are then expanded by growth inculture. The cells are then contacted with labeled target and sorted byFACS to isolate cells displaying AAs which fail to bind labeled targetin the absence of enzyme/reducing agent. These steps represent one cycleof the screening procedure. The cells can then be subjected toadditional cycles by expansion by growth in culture and again subjectingthe culture to all or part of the screening steps.

Library screening and sorting can also be initiated by first selectingfor candidates that do not bind labeled target in the absence ofenzyme/reduction agent treatment (i.e., do not bind target when notcleaved/reduced).

Example 3: In Vitro Screening of Modified Antibodies to DetermineMasking Efficiency of the MM

In order to screen modified antibodies and AAs that exhibit optimalcharacteristics when masked, for example, only 10% of binding to thetarget when in a masked state and in the presence of target, ABs coupledto different MMs or ABs coupled to the same MMs at different points ofattachment, or ABs coupled to the same MM via linkers of differentlengths and/or sequences are generated.

The masking efficiency of MMs can be determined by the affinity of theMM for AB and the spatial relationship of the MM relative to the bindinginterface of the AB to its target. Discovery of an effective MM is basedon affinity and as well optionally an empirical measure of maskingefficiency. The time-dependent target displacement of the MM in themodified antibody or AA can be measured to optimize and select for MMs.A immunoabsorbant Target Displacement Assay (TDA) is described hereinfor the discovery and validation of efficiently masked antibodies

In the TDA assay, the ability of an MM to inhibit AB binding to itstarget at therapeutically relevant concentrations and times is measured.The assay allows for measurement of the time-dependent targetdisplacement of the MM.

Briefly the antibody target is adsorbed to the wells of an ELISA plateovernight a about 4° C. The plate is blocked by addition of about 150 μl2% non-fat dry milk (NFDM) in PBS, about 0.5% (v/v) Tween20 (PBST), andincubation at room temperature for about 1 hour. The plate is thenwashed about three times with PBST. About 50 μl superblock is added(Thermo Scientific) and supplemented with protease inhibitors (Complete,Roche). About 50 μl of an AB coupled to a MM is dissolved in superblockwith protease inhibitors (Complete, Roch) and incubated at about 37° C.for different periods of time. The plate is washed about three timeswith PBST. About 100 ml of anti-huIgG-HRP is added in about 2% NFDM/PBSTand incubated at room temperature for about 1 hour. The plate is washedabout four times with PBST and about twice with PBS. The assay isdeveloped using TMB (Thermo Scientific) as per manufacturer'sdirections.

Example 4: AAs Comprising an scFv as the AB

Examples of AAs comprising an anti-Jagged1 scFv are described herein.These AAs are inactive (masked) under normal conditions due to theattached MM. When the scFv reaches the site of disease, however, adisease-specific enzyme such as ADAM17 will cleave a substrate linkerconnecting the peptide inhibitor to the scFv allowing it to bind toJagged1. Bacterial cell surface display is used to find suitable MMs forthe anti-Jagged1 scFv. In this example, selected MMs are combined withan enzyme substrate to be used as a trigger to create a scFv constructthat becomes competent for targeted binding after protease activation.

Construction of Protease Activated Antibody

Genes encoding AAs comprising a Jagged1 antibody in single-chain formare produced by overlap extension PCR or total gene synthesis andligated into a similarly digested expression plasmid or any othersuitable bacterial, yeast, or mammalian expression vector familiar toone skilled in the art. Full length antibodies can be alternativelyproduced using commercially available expression vectors incorporatinghalf-life extending moieties (e.g. the Fc region of an IgG, serumalbumin, or transferrin) and methods familiar to one skilled in the art.The expression plasmid is then transformed or transfected into anappropriate expression host such as BL21 for E. coli or HEK293t cells.Single chain antibodies are harvested from overnight cultures using aPeriplasmic fraction extraction kit (Pierce), and purified byimmobilized metal ion affinity chromatography, and by size exclusionchromatography.

Assay for Antibody Switching Activity In Vitro

Aliquots of protease-activated antibodies, at a concentration of 1 pM-1μM are incubated in a buffered aqueous solution separately with 0 and 50nM enzyme for 3 hrs. The reaction mixtures are then assayed for bindingusing ELISA or surface Plasmon resonance with immobilized antigenJagged1. An increase in binding activity for the AA after proteasetreatment is indicated by an increase in resonance units when usingBIAcore™ SPR instrumentation. The change in apparent dissociationconstant (K_(d)) as a result of cleavage can then be calculatedaccording the instrument manufacturer's instructions (BIAcore, GEHealthcare).

Example 5: Cloning of the Anti-VEGF scFv AB

In this and following examples an AA containing a masked MMP-9 cleavableanti-VEGF scFv (target=VEGF; AB=anti-VEGF single chain Fv) wasconstructed. As a first step in the production of such an AA, constructscontaining an anti-VEGF scFv were generated (the AB). An anti-VEGF scFvAB (V_(L)-linker L-V_(H)) was designed from the published sequence ofranibizumab (Genentech, Chen, Y., Wiesmann, C., Fuh, G., Li, B.,Christinger, H., McKay, P., de Vos, A. M., Lowman, H. B. (1999)Selection and Analysis of an Optimized Anti-VEGF Antibody: CrystalStructure of an Affinity-matured Fab in Complex with Antigen J. Mol.Biol. 293, 865-881) and synthesized by Codon Devices (Cambridge, Mass.).

Ranibizumab is a monoclonal antibody Fab fragment derived from the sameparent murine antibody as bevacizumab (Presta L G, Chen H, O'Connor S J,et al Humanization of an anti-vascular endothelial growth factormonoclonal antibody for the therapy of solid tumors and other disorders.Cancer Res, 57: 4593-9, 1997). It is smaller than the parent moleculeand has been affinity matured to provide stronger binding to VEGF-A.Ranibizumab binds to and inhibits all subtypes of vascular endothelialgrowth factor A (VEGF-A). A His6 tag (SEQ ID NO: 48) at the N-terminusand a TEV protease cleavage site were included in the design. The TEVprotease is a protease isolated from tobacco etch virus, is veryspecific, and is used to separate fusion proteins followingpurification. The anti-VEGF scFv nucleotide and amino acid sequences areprovided below in Tables 7 and 8.

TABLE 7 anti-VEGF scFv AB nucleotide sequencegatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcg (SEQ ID NO: 49)

TABLE 8 anti-VEGF scFv AB amino acid sequenceDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVS (SEQ ID NO: 50)

Example 6: Screening and Identification of MMs for Anti-VEGF scFv

Ranibizumab was used to screen a pooled random peptide library,consisting of peptides that are X₁₅ (8.3×10⁹), X₄CX₇CX₄ (3.6×10⁹), orX₁₂CX₃ (1.1×10⁹), where X is any amino acid and the number representsthe total diversity of the library. The total diversity of the pooledlibrary was 1.3×10¹⁰. The screening consisted of one round of MACS andtwo rounds of FACS sorting. In the first round MACS screen, 1×10¹¹ cellswere probed with 150 nM biotinylated-ranibizumab, and 5.5×10⁷ bindingcells were isolated. In the first FACS screen, positive cells isolatedin the MACS screen were probed with 500 nM biotinylated-ranibizumab, andvisualized with neutrAvidin-PE (Molecular Probes, Eugene, Oreg.). Thesecond and third rounds of FACS selections were done with 500 nM andthen 100 nM Alexa-labeled ranibizumab in the presence of 20 uM IgG.Individual clones were sequenced and subsequently verified for theirability to bind anti-VEGF scFv by FACS analysis. Amino acid sequences ofMMs for anti-VEGF scFv are provided in Table 9 below. (These sequenceswill hereafter be referred to as 283 MM, 292 MM, 306 MM, etc.)

TABLE 9 MMs for anti-VEGF scFv JS283 ATAVWNSMVKQSCYMQG (SEQ ID NO: 51)JS292 GHGMCYTILEDHCDRVR (SEQ ID NO: 52) JS306 PCSEWQSMVQPRCYYGG (SEQ IDNO: 53) JS308 NVECCQNYNLWNCCGGR (SEQ ID NO: 54) JS311 VHAWEQLVIQELYHC(SEQ ID NO: 55) JS313 GVGLCYTILEQWCEMGR (SEQ ID NO: 56) JS314RPPCCRDYSILECCKSD (SEQ ID NO: 57) JS315 GAMACYNIFEYWCSAMK (SEQ ID NO:58)

Example 7: Construction of the AA: MMP-9 Cleavable, Masked-Anti-VEGFscFv Vectors

A CM (substrate for MMP-9) was fused to the masked anti-VEGF scFvconstruct to provide a cleavable, masked AA. An exemplary construct isprovided in FIG. 4. Several exemplary AA constructs and sequencescontaining different CMs are described in great detail below. Primersutilized for construction of the exemplary constructs are represented inTable 10 below.

TABLE 10 Primers utilized for construction of MMP-9 Cleavable,masked-anti-VEGF scFv CX02335′gaattcatgggccatcaccatcaccatcacggtgggg3′ (SEQ ID NO: 59) CX02495′gtgagtaagcttttattacgacactgtaaccagagtaccctgg3′ (SEQ ID NO: 60) CX02705′gtggcatgtgcacttggccaccttggcccactcgagctggccagactggccctgaaaatacagattttccc3′(SEQ ID NO: 61) CX02715′gagtgggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcc3′(SEQ ID NO: 62) CX02885′ ttcgagctcgaacaacaacaacaataacaataacaacaac3′ (SEQ ID NO: 63) CX02895′ gctttcaccgcaggtacttccgtagctggccagtctggcc3′ (SEQ ID NO: 64) CX02905′ cgctccatgggccaccttggccgctgccaccagaaccgcc3′ (SEQ ID NO: 65) CX03085′ gcccagccggccatggccggccagtctggccagctcgagt3′ (SEQ ID NO: 66) CX03105′ ccagtgccaagcttttagtggtgatggtgatgatgcgacactgtaaccagagtaccctggcc3′ (SEQID NO: 67) CX0312 5′cttgtcacgaattcgggccagtctggccagctcgagt3′ (SEQ ID NO:68) CXO314 5′cagatctaaccatggcgccgctaccgcccgacactgtaaccagagtaccctg3′ (SEQID NO: 69)Cloning and Expression of the AA: A MMP-9 Cleavable, Masked Anti-VEGFscFv as a MBP Fusion

Cloning:

A MBP:anti-VEGF scFv AB fusion was cloned. The MBP (maltose bindingprotein) expresses well in E. coli, as a fusion protein, and can bepurified on a maltose column, a method well known in the art to makefusion proteins. In this example, the MBP was used to separate themasked scFv. The His6 tagged (SEQ ID NO: 48) Anti-VEGF scFv AB wascloned into the pMal-c4x vector (NEB) as a C-terminal fusion with MBPusing the EcoRI and HindIII restriction sites in the multiple cloningsite (MCS). The primers CX0233 and CX0249 (Table 10) were used toamplify the Anti-VEGF scFv AB and introduce the EcoRI and HindIII sites,respectively.

The accepting vector for the AA (the peptide MM, the anti-VEGF scFv ABand the MMP-9 CM) was synthesized using polymerase chain reaction (PCR)with the overlapping primers CX0271 and CX0270 which placed the cloningsite for the peptide MM's, linker sequences, and MMP-9 CM protease sitebetween the TEV protease site and the anti-VEGF scFv AB. The primersCX0271 and CX0249 (Table 10) were used to amplify the C-terminal portionof the construct, while the primers CX0270 and CX0288 (Table 10) wereused to amplify the N-terminal portion. Products from both the abovereactions were combined for a final PCR reaction using the outsideprimers, CX0249 and CX0288 (Table 10), and cloned into the pMal vectoras an MBP fusion using the SacI and HindIII restriction sites.

TABLE 11 MBP/MM accepting site/MMP-9 CM/Anti-VEGF scFv AB vectornucleotide sequence atgggccatcaccatcaccatcacggtggggaaaatctgtattttcagggccagtctggccagctcgagtgggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggt tacagtgtcg (SEQ IDNO: 70)

The 306 MM and 314 MM (Table 9) were amplified from the ecpX displayvector using the primers CX0289 and CX0290 (Table 10), and directionallycloned into the N-terminally masked vector using the SfiI restrictionsites. The corresponding nucleotide and amino acid sequences areprovided in Table 12 below.

TABLE 12 306 or 314 MM/MMP-9 CM/Anti-VEGF scFv AB Sequences MBP/306MM/MMP-9 CM/Anti-VEGF scFv AB nucleotide sequenceatgggccatcaccatcaccatcacggtggggaaaatctgtattttcagggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggt actctggttacagtgtcg(SEQ ID NO: 71) MBP/306 MM/MMP-9 CM/Anti-VEGF scFv AB amino acidsequence MGHHHHHHGGENLYFQGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQG TLVTVS (SEQ ID NO:72) MBP/314 MM/MMP-9 CM/Anti-VEGF scFv AB nucleotide sequenceatgggccatcaccatcaccatcacggtggggaaaatctgtattttcagggccagtctggccagcggccgccgtgttgccgtgattatagtattttggagtgctgtaagagtgatggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccag ggtactctggttacagtgtcg(SEQ ID NO: 73) MBP/314 MM/MMP-9 CM/Anti-VEGF scFv AB amino acidsequence MGHHHHHHGGENLYFQGQSGQRPPCCRDYSILECCKSDGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQ GTLVTVS (SEQ ID NO:74)

Expression:

Expression of the MBP:AA fusions were conducted in a K12 TB1 strain ofE. coli An ampicillin-resistant colony containing the desired constructwas used to inoculate a 5 ml overnight culture containing LB mediumsupplemented with 50 μg/mL Ampicillin. The entire overnight culture wasused to inoculate 500 mL of fresh LB medium supplemented with 50 μg/mLampicillin and 0.2% Glucose and allowed to grow at 37° C. shaking at 250rpm until an O.D. of 0.5 was reached. Isopropylthio-β-D-galactosidasewas then added to a final concentration of 0.3 mM and the culture wasallowed to grow for a further 3 hrs under the same conditions afterwhich the cells were harvested by centrifugation at 3000×g. Inclusionbodies were purified using standard methods. Briefly, 10 mls of BPER IIcell lysis reagent (Pierce). Insoluble material was collected bycentrifugation at 14,000×g and the soluble proteins were discarded. Theinsoluble materials were resuspended in 5 mls BPER II supplemented withlmg/mL lysozyme and incubated on ice for 10 minutes after which 5 mls ofBPER II diluted in water 1:20 was added and the samples were spun at14,000×g. The supernatant was removed and the pellets were wash twice in1:20 BPERII. The purified inclusion bodies were solubilized in PB S 8 MUrea, 10 mM BME, pH 7.4.

The MBP fusion proteins were diluted to a concentration of approximately1 mg/mL and refolded using a stepwise dialysis in PBS pH 7.4 from 8 to 0M urea through 6, 4, 2, 0.5, and 0 M urea. At the 4, 2, and 0.5 M Ureasteps 0.2 M Arginine, 2 mM reduced Glutathione, and 0.5 mM oxidizedglutathione was added. The 0M Urea dialysis included 0.2 M Arginine.After removal of the urea, the proteins were dialyzed against 0.05 MArginine followed by and extensive dialysis against PBS pH 7.4. Alldialysis were conducted at 4° C. overnight. To remove aggregates, eachprotein was subjected to size exclusion chromatography on a sephacrylS-200 column. Fractions containing the correctly folded proteins wereconcentrated using an Amicon Ultra centrifugal filter.

Cloning and Expression of the AA: A MMP-9 Cleavable, Masked Anti-VEGFscFv CHis Tag

Cloning:

The primers CX0308 and CX0310 (Table 10) were used to amplify and add aNcoI restriction site to the 5′ end and a HindIII restriction site andHis6 tag (SEQ ID NO: 48) to the 3′ end, respectively, of the (MMaccepting site/MMP-9 CM/VEGFscFv AB) vector which was subsequentlycloned into a vector containing the pelB signal peptide. Anti-VEGF scFvMMs were cloned as previously described. The corresponding nucleotideand amino acid sequences are provided in Table 13.

TABLE 13 306 or 314 MM/MMP-9 CM/anti-VEGF scFv CHis AB Sequences 306MM/MMP-9 CM/anti-VEGF scFv CHis AB nucleotide sequenceggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgcatcatcaccatcaccac (SEQ ID NO: 75) 306 MM/MMP-9CM/anti-VEGF scFv CHis AB amino acid sequenceGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSHHHHHH (SEQ ID NO: 76) 314MM/MMP-9 CM/anti-VEGF scFv CHis AB nucleotide sequenceggccagtctggccagcggccgccgtgttgccgtgattatagtattttggagtgctgtaagagtgatggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgcatcatcaccatcaccactaa (SEQ ID NO: 77) 314MM/MMP-9 CM/anti-VEGF scFv CHis AB amino acid sequenceGQSGQRPPCCRDYSILECCKSDGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSHHHHHH (SEQ ID NO: 78)

Expression:

Expression of the Anti-VEGF scFv His AAs was conducted in a K12 TB 1strain of E. coli An ampicillin-resistant colony containing the desiredconstruct was used to inoculate a 5 ml overnight culture containing LBmedium supplemented with 50 μg/mL Ampicillin. 2.5 ml of overnightculture was used to inoculate 250 mL of fresh LB medium supplementedwith 50 μg/mL ampicillin and 0.2% Glucose and allowed to grow at 37° C.shaking at 250 rpm until an O.D. of 1.0 was reached.Isopropylthio-β-D-galactosidas was then added to a final concentrationof 0.3 mM and the culture was allowed to grow for a further 5 hrs at 30°C. after which the cells were harvested by centrifugation at 3000×g. Theperiplasmic fraction was immediately purified using the lysozyme/osmoticshock method. Briefly, the cell pellet was resuspended in 3 mLs of 50 mMTris, 200 mM NaCl, 10 mM EDTA, 20% Sucrose, pH 7.4 and 2 uL/mL ready-uselysozyme solution was added. After a 15 min. incubation on ice, 1.5volumes of water (4.5 mLs) was added and the cells were incubated foranother 15 min. on ice. The soluble periplasmic fraction was recoveredby centrifugation at 14,000×g.

The Anti-VEGF scFv His proteins were partially purified using Ni-NTAresin. Crude periplasmic extracts were loaded onto 0.5 ml of Ni-NTAresin and washed with 50 mM phosphate, 300 mM NaCl, pH 7.4. His taggedproteins were eluted with 50 mM phosphate, 300 mM NaCl, 200 mMImidizale, pH 6.0. Proteins were concentrated to approximately 600 μLand buffer exchanged into PBS using Amicon Ultra centrifugalconcentrators.

Cloning and Expression of the AA: A MMP-9 Cleavable, Masked Anti-VEGFscFv as Human Fc Fusion

Cloning: The primers CX0312 and CXO314 (Table 10) were used to amplifythe sequence encoding MMP-9 CM/Anti-VEGF scFv. The primers also includedsequences for a 5′ EcoRI restriction site and a 3′ NcoI restriction siteand linker sequence. Cutting the PCR amplified sequence with EcoRI andNcoI and subsequent cloning into the pFUSE-hIgG1-Fc2 vector generatedvectors for the expression of Fc fusion proteins. Anti-VEGF scFv AB MMswere inserted into these vectors as previously described. Constructscontaining 306 MM, 313 MM, 314 MM, 315 MM, a non-binding MM (100 MM), aswell as no MM were constructed and sequences verified. The correspondingnucleotide and amino acid sequences are provided below in Table 14.

TABLE 14 306 MM/MMP-9 CM/anti-VEGF scFv-Fc AB sequences 306 MM/MMP-9CM/anti-VEGF scFv-Fc AB nucleotide sequenceggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgggcggtagcggcgccatggttagatctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID NO: 79) 306 MM/MMP-9 CM/anti-VEGFscFv-Fc AB amino acid sequenceGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK (SEQ ID NO: 80) 314 MM/MMP-9 CM/anti-VEGFscFv-Fc AB nucleotide sequenceggccagtctggccagcggccgccgtgttgccgtgattatagtattttggagtgctgtaagagtgatggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaaggggggtggaggcagcgggggaggtggctcaggcggtggagggtctggcgaggtccagctggtagaaagcgggggcggactggtccaaccgggcggatccctgcgtctgagctgcgcggcctcgggttacgactttactcactacggaatgaactgggttcgccaagcccctggtaaaggtctggaatgggtcggatggattaatacatacactggagaacctacttatgctgctgatttcaaacgtcgctttactttctctctggatacaagtaagtcaaccgcctatctgcaaatgaacagcctgcgtgcagaggacacggctgtgtactattgtgcgaaatatccttattattatggaacttcccactggtatttcgatgtatggggccagggtactctggttacagtgtcgggcggtagcggcgccatggttagatctgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggaggagatgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgagggtctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID NO: 81) 314 MM/MMP-9CM/anti-VEGF scFv-Fc AB amino acid sequenceGQSGQRPPCCRDYSILECCKSDGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK (SEQ ID NO: 82)

Expression:

10 μg of expression vectors for 306 MM/MMP-9 CM/anti-VEGFscFv-Fc, 314MM/MMP-9 CM/anti-VEGFscFv-Fc or anti-VEGFscFv-Fc were introduced into10⁷ HEK-293 freestyle cells (Invitrogen, CA) by transfection usingtransfectamine 2000 as per manufacturer's protocol (Invitrogen, CA). Thetransfected cells were incubated for an additional 72 hours. Afterincubation, the conditioned media was harvested and cleared of cells anddebris by centrifugation. The conditioned media was assayed for activityby ELISA.

Example 8: Measurement of the Activation of a Masked MMP-9 Cleavable AA

To measure the activation of the masked MMP-9 cleavable anti-VEGF AAs byMMP-9, 100 μl of a 2 μg/ml PBS solution of VEGF was added to microwells(96 Well Easy Wash; Corning) and incubated overnight at 4° C. Wells werethen blocked for 3×15 minute with 300 μL Superblock (Pierce). Onehundred microliters of an AA (see below for details pertaining to eachconstruct), treated or untreated with MMP-9, were then added to wells inPBST, 10% Superblock and incubated at room temperature (RT) for 1 hr.All wash steps were done three times and performed with 300 μl PBST. Onehundred microliters of secondary detection reagent were then added andallowed to incubate at RT for 1 hr. Detection of HRP was completed using100 μl of TMB one (Pierce) solution. The reaction was stopped with 100μL of iN HCL and the absorbance was measured at 450 nM.

ELISA Assay of an AA Construct Containing: MBP/MM/MMP-9 CM/Anti-VEGFscFv AB

Two hundred microliters of biotinylated AA in MMP-9 digestion buffer (50mM Tris, 2 mM CaCl₂, 20 mM NaCl, 100 μM ZnCl₂, pH 6.8) at aconcentration of 200 nM was digested with 20 U TEV protease overnight at4° C. to remove the MBP fusion partner. Samples were then incubated for3 hrs with or without ˜3 U of MMP-9 at 37° C., diluted 1:1 to a finalconcentration of 100 nM in PBST, 10% Superblock, and added to the ELISAwells. Detection of the AA was achieved with an Avidin-HRP conjugate ata dilution of 1:7500. MMP-9 activation of MMP-9 cleavable maskedMBP:anti-VEGF scFv AA is presented in FIG. 5.

ELISA Assay of an AA Construct Containing: MM/MMP-9 CM/Anti-VEGF scFvhis

Crude periplasmic extracts dialyzed in MMP-9 digestion buffer (150 μL)were incubated with or without ˜3 U of MMP-9 for 3 hrs at 37° C. Sampleswere then diluted to 400 μL with PBST, 10% Superblock and added to theELISA wells. Detection of the AA was achieved using an Anti-His6 (SEQ IDNO: 48)—HRP conjugate at a dilution of 1:5000. MMP-9 activation of MMP-9cleavable masked anti-VEGF scFv His AA is presented in FIG. 6.

ELISA Assay of an AA Construct Containing: MM/MMP-9 CM/Anti-VEGF scFv-Fc

Fifty microliters of HEK cell supernatant was added to 200 μL MMP-9digestion buffer and incubated with or without 19 U MMP-9 for 2 hrs at37° C. Samples were then diluted 1:1 in PBST, 10% Superblock and 100 μLwere added to the ELISA wells. Detection of the AA was achieved usingAnti-human Fc-HRP conjugate at a dilution of 1:2500. MMP-9 activation ofMMP-9 cleavable masked anti-VEGF scFv-Fc is presented in FIG. 7.

Purification and Assay of an AA Construct Containing: MM/MMP-9CM/Anti-VEGF scFv-Fc

Anti-VEGF scFv Fc AAs were purified using a Protein A columnchromatography. Briefly, 10 mLs of HEK cell supernatants were diluted1:1 with PBS and added to 0.5 mL Protein A resin pre-equilibrated inPBS. Columns were washed with 10 column volumes of PBS before elutingbound protein with 170 mM acetate, 300 mL NaCl pH. 2.5 and immediatleyneutralized 1 mL fractions with 200 μL of 2 M Tris pH 8.0. Fractionscontaining protein were then concentrated using Amicon Ultra centrifugalconcentrators. ELISA was conducted as with HEK cell supernatants. ELISAdata showing the MMP-9 dependent VEGF binding of Anti-VEGFscFv Fc AAconstructs with the MMs 306 and 314 that were purified using a Protein Acolumn are presented in FIG. 8.

Example 9: Target Displacement Assay for the Discovery and Validation ofEfficiently Masked Therapeutic Proteins

VEGF was adsorbed to the wells of a 96-well micro-titer plate, washedand blocked with milk protein. 25 ml of culture media containinganti-VEGF antibody or anti-VEGF AA's containing the MM JS306, was addedto the coated wells and incubated for 1, 2, 4, 8 or 24 hours. Followingincubation, the wells were washed and the extent of bound AA's wasmeasured by anti-huIgG immunodetection. FIG. 9 shows mask 306 cancompletely inhibit binding to VEGF at one hour; however, at 16hours, >50% of the 306-antiVEGF AA is bound to its antigen, VEGF. The306 mask, which binds to anti-VEGF antibody with an affinity of >600 nM,does not efficiently preclude binding to VEGF.

Example 10: Library Screening and Isolation of Anti-CTLA4 MMs

CTLA4 antibody masking moieties (MMs) were isolated from a combinatoriallibrary of 10¹⁰ random 15 mer peptides displayed on the surface of E.coli according to the method of Bessette et al (Bessette, P. H., Rice,J. J and Daugherty, P. S. Rapid isolation of high-affinity proteinbinding peptides using bacterial display. Protein Eng. Design &Selection. 17:10,731-739, 2004). Biotinylated mouse anti-CTLA4 antibody(clone UC4 F10-11, 25 nM) was incubated with the library andantibody-bound bacteria expressing putative binding peptides weremagnetically sorted from non-binders using streptavidin-coated magneticnanobeads. Subsequent rounds of enrichment were carried out using FACS.For the initial round of FACS, bacteria were sorted using biotinylatedtarget (5 nM) and secondary labeling step with streptavidiinphycoerythrin. In subsequent rounds of FACS, sorting was performed withDylight labeled antibody and the concentration of target was reduced (1nM, then 0.1 nM) to avoid the avidity effects of the secondary labelingstep and select for the highest affinity binders. One round of MACS andthree rounds of FACS resulted in a pool of binders from which individualclones were sequenced. Relative affinity and off-rate screening ofindividual clones were performed using a ficin digested Dylight-labeledFab antibody fragment to reduce avidity effects of the bivalent antibodydue to the expression of multiple peptides on the bacterial surface. Asan additional test of target specificity, individual clones werescreened for binding in the presence of 20 μM E. Coli depleted IgG as acompetitor. Amino acid and nucleotide sequences of the 4 clones chosenfor MM optimization are shown in Table 15. These sequences willinterchangeably referred to as 115 MM, 184 MM, 182 MM, and 175 MM. MMcandidates with a range of off-rates were chosen, to determine theeffects of off-rates on MM dissociation after cleavage. An MM that didnot bind anti-CTLA4 was used as a negative control.

TABLE 15 Amino acid and nucleotide sequences for MMs that maskanti-CTLA4 KK115 MM M I L L C A A G R T W V E A C A N G R (SEQ ID NO:84) ATGATTTTGTTGTGCGCGGCGGGTCGGACGTGGGTGGAGGCTTGCGCTAA TGGTAGG (SEQ IDNO: 83) KK184 MM A E R L C A W A G R F C G S (SEQ ID NO: 86)GCTGAGCGGTTGTGCGCGTGGGCGGGGCGGTTCTGTGGCAGC (SEQ ID NO: 85) KK182 MM W AD V M P G S G V L P W T S (SEQ ID NO: 88)TGGGCGGATGTTATGCCTGGGTCGGGTGTGTTGCCGTGGACGTCG (SEQ ID NO: 87) KK175 MM SD G R M G S L E L C A L W G R F C G S (SEQ ID NO: 90)AGTGATGGTCGTATGGGGAGTTTGGAGCTTTGTGCGTTGTGGGGGCGGTT CTGTGGCAGC (SEQ IDNO: 89) Negative control (does not bind anti-CTLA4) P C S E W Q S M V QP R C Y Y (SEQ ID NO: 92) CCGTGTTCTGAGTGGCAGTCGATGGTGCAGCCGCGTTGCTATTAT(SEQ ID NO: 91)

Example 11: Cloning of Anti-CTLA4 scFv

Anti-CTLA4 ScFv was cloned from the HB304 hybridoma cell line (AmericanType Culture Collection) secreting UC4F10-11 hamster anti-mouse CTLA4antibody according to the method of Gilliland et al. (Gilliland L. K.,N. A. Norris, H. Marquardt, T. T. Tsu, M. S. Hayden, M. G. Neubauer, D.E. Yelton, R. S. Mittler, and J. A. Ledbetter. Rapid and reliablecloning of antibody variable regions and generation of recombinantsingle chain antibody fragments. Tissue Antigens 47:1, 1-20, 1996). Adetailed version of this protocol can be found at the Institute ofBiomedical Sciences (IBMS) at Academia Sinica in Taipei, Taiwan website.In brief, total RNA was isolated from hybridomas using the RNeasy totalRNA isolation kit (Qiagen). The primers IgK1 (gtyttrtgngtnacytcrca (SEQID NO: 93)) and IgH (acdatyttyttrtcnacyttngt (SEQ ID NO: 94)) (Gillilandet al. referenced above) were used for first strand synthesis of thevariable light and heavy chains, respectively. A poly G tail was addedwith terminal transferase, followed by PCR using the 5′ ANCTAIL primer(Gilliland et al. referenced above)(cgtcgatgagctctagaattcgcatgtgcaagtccgatggtcccccccccccccc (SEQ ID NO:95)) containing EcoRI, SacI and XbaI sites for both light and heavychains (poly G tail specific) and the 3′ HBS-hIgK(cgtcatgtcgacggatccaagcttacyttccayttnacrttdatrtc (SEQ ID NO: 96)) andHBS-hIgH (cgtcatgtcgacggatccaagcttrcangcnggngcnarnggrtanac (SEQ ID NO:97)) derived from mouse antibody constant region sequences andcontaining HindIII, BamHI and SalI sites for light and heavy chainamplification, respectively (Gilliland et al. referenced above).Constructs and vector were digested with HindIII and SacI, ligated andtransformed into E. Coli. Individual colonies were sequenced and thecorrect sequences for V_(L) and V_(H) (Tables 16 and 17 respectively)were confirmed by comparison with existing mouse and hamster antibodies.The leader sequences, as described for anti-CTLA4 in the presentedsequence is also commonly called a signal sequence or secretion leadersequence and is the amino acid sequence that directs secretion of theantibody. This sequence is cleaved off, by the cell, during secretionand is not included in the mature protein. Additionally, the same scFvcloned by Tuve et al (Tuve, S. Chen, B. M., Liu, Y., Cheng, T-L., Toure,P., Sow, P. S., Feng, Q., Kiviat, N., Strauss, R., Ni, S., Li, Z.,Roffler, S. R. and Lieber, A. Combination of Tumor Site-LocatedCTL-Associated Antigen-4 Blockade and Systemic Regulatory T-CellDepletion Induces Tumor Destructive Immune Responses. Cancer Res. 67:12,5929-5939, 2007) was identical to sequences presented here.

TABLE 16 Hamster anti-mouse CTLA4 V_(L)

M E S H I H V F M S L F L W V S G S C A D I M M T Q SP S S L S V S A G E K A T I S C K S S Q S L F N S N AK T N Y L N W Y L Q K P G Q S P K L L I Y Y A S TR H T G V P D R F R G S G T D F T L T I S S V Q D ED L A F Y Y C Q Q W Y D Y P Y T F G A G T K V E I K (SEQ ID NO: 98)atggaatcacatatccatgtcttcatgtccttgttcctttgggtgtctggttcctgtgcagacatcatgatgaccagtctccttcatccctgagtgtgtcagcgggagagaagccactatcagctgcaagtccagtcagagtcttttcaacagtaacgccaaaacgaactacttgaactggtatttgcagaaaccagggcagtctcctaactgctgatctattatgcatccactaggcatactggggtccctgatcgcttcagaggcagtggatctgggacggatttcactctcaccatcagcagtgtccaggatgaagacctggcattttattactgtcagcagtggtatgactacccatacacgttcggagctgggaccaaggtggaaatcaaa (SEQ ID NO: 99)

TABLE 17 Hamster anti mouse CTLA4 V_(H)

K M R L L G L L Y L V T A L P G V L S Q I Q L Q E SG P G L V N P S Q S L S L S C S V T G Y S I T S G Y GW N W I R Q F P G Q K V E W M G F I Y Y E G S T YY N P S I K S R I S I T R D T S K N Q F F L Q V N S VT T E D T A T Y Y C A R Q T G Y F D Y W G Q G T M VT V S S (SEQ ID NO: 100)aagatgagactgttgggtettetgtacetggtgacageeettectggtgtectgteccagatecagetteaggagteaggacetggeetggtgaacceeteacaateactgtecetetettgetetgteactggttactecateaccagtggttatggatggaactggateaggeagtteccagggcagaaggtggagtggatgggattcatatattatgagggtagcacctactacaacccttccatcaagagccgcatctccatcaccagagacacategaagaaccagttettectgeaggtgaattetgtgaccactgaggacacagecacatattactgtgegagacaaactgggtactttgattactggggccaaggaaccatggtcaccgtctcctca (SEQ ID NO: 101)

Example 12: Construction of the Anti-CTLA4 scFv with MMs and CMs

To determine the optimal orientation of the anti-CTLA4 scFv forexpression and function, primers were designed to PCR amplify thevariable light and heavy chains individually, with half of a (GGGS) 3linker (SEQ ID NO: 102) at either the N- or C-terminus for a subsequent‘splicing by overlapping extension’ PCR (SOE-PCR; Horton, R. M., Hunt,H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. (1989) Engineeringhybrid genes without the use of restriction enzymes: gene splicing byoverlap extension. Gene 77, 61-68) with either V_(H) or V_(L) at theN-terminus. An NdeI restriction site was engineered at the N-terminus togenerate a start codon in frame at the beginning of the nucleotidesequence and a His tag and stop codon were added to the C-terminus.Light and heavy chains were then joined via sewing PCR using the outerprimers to generate ScFvs in both V_(H)V_(L) and V_(L)V_(H) (FIG. 10).Primers are shown below in Table 18.

TABLE 18 Primers to generate scFvs V_(H)V_(L) and V_(L)V_(H) VL for1caaggaccatagcatatggacatcatgatgacccagtct (SEQ ID NO: 103) VL linkeracttccgcctccacctgatccaccaccacctttgatttccaccttggtcc (SEQ ID NO: 104) rev1linker VH ggatcaggtggaggcggaagtggaggtggcggttcccagatccagcttcaggagtcagga(SEQ ID NO: 105) for2 VH his rev2ggccggatccaagcttttagtggtgatggtgatgatgtgaggagacggtgaccatggttcc (SEQ IDNO: 106) VH for3 acaaggaccatagcatatgcagatccagcttcaggagtca (SEQ ID NO:107) VH linker acttccgcctccacctgatccaccaccacctgaggagacggtgaccatggttcc(SEQ ID NO: 108) rev3 linker VLggtggatcaggtggaggcggaagtggaggtggcggttccgacatcatgatgacccagtctcct (SEQ IDNO: 109) for4 VL his rev4cggccggatccaagcttttagtggtgatggtgatgatgtttgatttccaccttggtcccagc (SEQ IDNO: 110)

Next, a set of overlapping primers were designed to add sfi and xholsites for MM cloning followed by the MMP-9 cleavage sequence and (GGS)2linker (SEQ ID NO: 111) on the N-terminus of the ScFv constructs. Theseprimers are presented in Table 19 and shown schematically in FIG. 10.

TABLE 19 Primers MM and CM cloning for 1c linkergccagtctggccggtagggctcgagcggccaagtgcacatgccactgggcttcctgggtc (SEQ ID NO:112) for 1d linkergccactgggcttcctgggtccgggtggaagcggcggctcagacatcatgatgacccagtc (SEQ ID NO:113) VL for 1e linkergccactgggcttcctgggtccgggtggaagcggcggctcacagatccagcttcaggagtca (SEQ IDNO: 114) VH for 1a ttcaccaacaaggaccatagcatatgggccagtctggccggtagggc (SEQID NO: 115) VH his rev2ggccggatccaagcttttagtggtgatggtgatgatgtgaggagacggtgaccatggttcc (SEQ IDNO: 116) VH linker rev3acttccgcctccacctgatccaccaccacctgaggagacggtgaccatggttcc (SEQ ID NO: 117)

Linker containing ScFvs were PCR amplified, digested with Nde1 and EcoR1(an internal restriction site in V_(H)) and gel purified. The PCRfragments were ligated into the vectors and transformed into E. coli Thenucleotide and amino acid sequences are presented in Table 20.

TABLE 20 Sequence of MM linker-CM-anti-CTLA4 scFv linker Amino acidsequence: (-------MM Linker-------)(---------------------CM-------------------) (---scFv Linker---) G G S GG S G G S S G Q V H M P L G F L G P G G S G G S (SEQ ID NO: 118)Nucleotide sequence:GGCGGTTCTGGTGGCAGCGGTGGCTCGAGCGGCCAAGTGCACATGCCACTGGGCTTCCTGGGTCCGGGTGGAAGCGGCGGCTCA (SEQ ID NO: 119)

MM sequences were PCR amplified, digested at sfi1 and xho1 sites,ligated into linker anti-CTLA4 scFv constructs, transformed into E. coliand sequenced. The complete nucleotide and amino acid sequences of theMM115-CM-AB are shown below in Tables 21 and 22 respectively.

TABLE 21 Amino acid sequence of MM115-anti-CTLA4 ScFv AB M I L L C A A GR T W V E A C A N G R G G S G G S G G S S G Q V H M P L G F L G P G G SG G S Q I Q L Q E S G P G L V N P S Q S L S L S C S V T G Y S I T S G YG W N W I R Q F P G Q K V E W M G F I Y Y E G S T Y Y N P S I K S R I SI T R D T S K N Q F F L Q V N S V T T E D T A T Y Y C A R Q T G Y F D YW G Q G T M V T V S S G G G G S G G G G S G G G G S D I M M T Q S P S SL S V S A G E K A T I S C K S S Q S L F N S N A K T N Y L N W Y L Q K PG Q S P K L L I Y Y A S T R H T G V P D R F R G S G S G T D F T L T I SS V Q D E D L A F Y Y C Q Q W Y D Y P Y T F G A G T K V E I K (SEQ IDNO: 120)

TABLE 22 Nucleotide sequence of MM115-anti-CTLA4 ScFv ABatgattttgttgtgcgcggcgggtcggacgtgggtggaggcttgcgctaatggtaggggcggttctggtggcagcggtggctcgagcggccaagtgcacatgccactgggcttcctgggtccgggtggaagcggcggctcacagatccagcttcaggagtcaggacctggcctggtgaacccctcacaatcactgtccctctcttgctctgtcactggttactccatcaccagtggttatggatggaactggatcaggcagttcccagggcagaaggtggagtggatgggattcatatattatgagggtagcacctactacaacccttccatcaagagccgcatctccatcaccagagacacatcgaagaaccagttcttcctgcaggtgaattctgtgaccactgaggacacagccacatattactgtgcgagacaaactgggtactttgattactggggccaaggaaccatggtcaccgtctcctcaggtggtggtggatcaggtggaggcggaagtggaggtggcggttccgacatcatgatgacccagtctccttcatccctgagtgtgtcagcgggagagaaagccactatcagctgcaagtccagtcagagtcttttcaacagtaacgccaaaacgaactacttgaactggtatttgcagaaaccagggcagtctcctaaactgctgatctattatgcatccactaggcatactggggtccctgatcgcttcagaggcagtggatctgggacggatttcactctcaccatcagcagtgtccaggatgaagacctggcattttattactgtcagcagtggtatgactacccatacacgttcggagctgggaccaaggtggaaatcaaacatcatcaccatcaccactaa (SEQ ID NO: 121)

To generate MM-CM-anti-CTLA4 scFv-Fc fusions, the following primerslisted in Table 23 were designed to PCR amplify the constructs forcloning into the pfuse Fc vector via the in fusion system (Clontech).Plasmids were transformed into E. coli, and the sequence of individualclones was verified.

TABLE 23 Primers to generate MM-CM-anti-CTLA4 scFv-Fc fusionsHLCTLA4ScFv pFuse reverse tcagatctaaccatggctttgatttccaccttggtcc (SEQ IDNO: 122) LHCTLA4ScFv pFuse reverse tcagatctaaccatggctgaggagacggtgaccatgg(SEQ ID NO: 123) p115CTLA4 pfuse forwardcacttgtcacgaattcgatgattttgttgtgcgcggc (SEQ ID NO: 124) p182CTLA4 pfuseforward cacttgtcacgaattcgtgggcggatgttatgcctg (SEQ ID NO: 125) p184CTLA4pfuse forward cacttgtcacgaattcggctgagcggttgtgcgcgtg (SEQ ID NO: 126)p175CTLA4 pfuse forward cacttgtcacgaattcgagtgatggtcgtatggggag (SEQ IDNO: 127) pnegCTLA4 pfuse forward cacttgtcacgaattcgccgtgttctgagtggcagtcg(SEQ ID NO: 128)

Example 13: Expression and Assay of Masked/MMP-9/Anti-CTLA4 scFv-Fc inHEK-293 Cells

10 μg of expression vectors for p175CTLA4pfuse, p182CTLA4pfuse,p184CTLA4pfuse, p115CTLA4pfuse, or pnegCTLA4pfuse were introduced into10⁷ HEK-293 freestyle cells (Invitrogen) by transfection usingtransfectamine 2000 as per manufacturer's protocol (Invitrogen). Thetransfected cells were incubated for an additional 72 hours. Afterincubation the conditioned media was harvested and cleared of cells anddebris by centrifugation. The conditioned media was assayed for activityby ELISA as described below.

Fifty microliters of conditioned media from HEK-293 expressingMM175-anti-CTLA4 scFv, MM182-anti-CTLA4 scFv, MM184-anti-CTLA4 scFv,MM115-anti-CTLA4 scFv, or MMneg-anti-CTLA4 scFv was added to 200 μLMMP-9 digestion buffer and incubated with or without ˜19 U MMP-9 for 2hrs at 37° C. Samples were then diluted 1:1 in PBS, 4% non fat dry milk(NFDM) and assayed for binding activity by competition ELISA.

100 μl of 0.5 mg/ml solution of murine CTLA4-Fc fusion protein (R & Dsystems) in PBS was added to wells of 96 well Easy Wash plate (Corning)and incubated overnight at 4° C. Wells were then blocked for one hour atroom temperature (RT) with 100 μl of 2% non-fat dry milk (NFDM) in PBSand then washed 3× with PBS; 0.05% Tween-20 (PBST). 50 μl of conditionedmedia from cultures of transfected HEK-293 cells expressingMM175-anti-CTLA4 scFv, MM182-anti-CTLA4 scFv, MM184-anti-CTLA4 scFv,MM115-anti-CTLA4 scFv, or MMneg-anti-CTLA4 scFv that had previously beenuntreated or treated with MMP-9, were added to wells and incubated RTfor 15 minutes. Following incubation, 50 μl of PBS containing 0.5 μg/mlbiotinylated murine B71-Fc (R & D systems) was added to each well.Following a further incubation at RT of 30 minutes the wells were washed5× with 150 μl PBST. 100 μl of PBS containing 1:3000 dilution ofavidin-HRP was added and the plate incubated at RT for 45 minutes andthen washed 7× with 150 μl PBST. The ELISA was developed with 100 μl ofTMB (Pierce), stopped with 100 μL of iN HCL and the absorbance wasmeasured at 450 nM.

Example 14: Construction of an Anti-CTLA4

Tables 24 and 25 display nucleotide and amino acid sequences foranti-human CTLA-4 scFv, respectively. M13 bacteriophage capable ofbinding human CTLA were supplied (under contract, by Creative Biolabs,21 Brookhaven Blvd., Port Jefferson Station, N.Y. 11776). Phage wereproduced in E. coli TG-1 and purified by PEG; NaCl precipitation.

TABLE 24 anti-human CTLA4 scFv AB nucleotide sequencegaaattgtgttgacacagtctccaggcaccctgtctttgtctccaggggaaagagccaccctctcctgcagggccagtcagagtgttagcagcagctacttagcctggtaccagcagaaacctggccaggctcccaggctcctcatctatggtgcatccagcagggccactggcatcccagacaggttcagtggcagtgggtctgggacagacttcactctcaccatcagcagactggagcctgaagattttgcagtgtattactgtcagcagtatggtagctcaccgctcactttcggcggagggaccaaggtggaaatcaaacgttccggagggtcgaccataacttcgtataatgtatactatacgaagttatcctcgagcggtacccaggtgcagctggtgcagactgggggaggcgtggtccagcctgggaggtccctgagactctcctgtgcagcctctggatccacctttagcagctatgccatgagctgggtccgccaggctccagggaaggggctggagtgggtctcagctattagtggtagtggtggtagcacatactacgcagactccgtgaagggccggttcaccatctccagagacaattccaagaacacgctgtatctgcaaatgaacagcctgagagccgaggacacggccgtatattactgtgcgacaaactccctttactggtacttcgatctctggggccgtggcaccctggtcactgtctcttcagctagc (SEQ ID NO: 129)

TABLE 25 anti-human CTLA4 scFv AB amino acid sequenceEIVLTQSPGTLSLSPGERATLSCRASQSVSSSYLAWYQQKPGQAPRLLIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVYYCQQYGSSPLTFGGGGTKVEIKRSGGSTITSYNVYYTKLSSSGTQVQLVQTGGGVVQPGRSLRLSCAASGSTFSSYAMSWVRQAPGKGLEWVSAIAGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAVYYCATNSLYWYFDLWGRGTLVTVSSA S (SEQ ID NO: 130)

Phage ELISA Measurement of CTLA-4 Binding:

To measure the binding of anti-CTLA-4 scFv-C2, 100 μl of a 0.5 μg/mlHuman CTLA-4-IgG or murine CTLA-4-IgG (R&D Systems) in PBS was added tomicrowells (96 Well Easy Wash; Corning) and incubated overnight at 4° C.Wells were then blocked for 1 hour at room temperature (RT) with 150 μlof 2% non-fat dry milk (NFDM) in PBST (PBS, pH 7.4, 0.5% Tween-20). Thewells were then washed 3× with 300 μl PBST. Following washing 100 μl ofpurified anti-CTLA-4 scFv phage in PBST were added to triplicate wellsand incubated RT for 1 hr. The wells were then washed 3× with 300 μlPBST. One hundred microliters of anti-M13 HRP-conjugated antibody wasthen added and incubated at RT for 1 hr. Detection of HRP was completedusing 100 μl of TMB one (Pierce) solution. The reaction was stopped 100μl of iN HCL and the absorbance was measured at 450 nM. FIG. 19 showsthe binding of anti-CTLA4 scFv to both murine and human CTLA4.

AAs Comprising an IgG as the AB

Examples of AAs comprising an anti-EGFR and anti-VEGF in the human IgGare described in the following sections. These AAs are masked andinactive under normal conditions. When the AAs reach the diseasedtissue, they are cleaved by a disease-specific protease and can thenbind their target. Bacterial display is used to discover suitable MMsfor the anti-EGFR and anti-VEGF antibodies. In, these examples, selectedMMs are combined with an enzyme substrate to be used as a trigger tocreate AAs that become competent for specific binding to targetfollowing protease activation. Furthermore, bacterial display is used toalter the discovered peptides to increase affinity for the ABs andenhance the inhibition of targeted binding in the un-cleaved state. Theincreased MM affinity and enhanced inhibition is important forappropriate AA function.

Example 15: Construction of an Anti-VEGF I2G AA

Construction of the Anti-VEGF IgG Antibody

The anti-VEGF light chain variable region was PCR amplified with primersCX0311 and CX0702 using the anti-VEGF mmp-9 306 scFv (described above)as template and then cloned into the pFIL2-CL-hk vector using the EcoRIand BsiWI restriction sites (pFIL2-VEGF-Lc). The 306 mmp-9 light chainwas PCR amplified with primers CX0325 and CX0702 using the anti-VEGFmmp-9 scFv as template and cloned as above (pFIL2-306mVEGF-Lc). Theanti-VEGF heavy chain variable regions were PCR amplified using primersCX0700 and CX0701 using the 306 MM/MMP-9 CM/anti-VEGFscFv (describedabove) as template and cloned into the pFIL-CHIg-hG1 vector using theEcoRI and NheI restriction sites (pFIL-VEGF-Hc). The primers areprovided below in Table 26.

TABLE 26 Primers for Construction of an anti-VEGF IgG antibody CX0311cttgtcacgaattcggatattcaactgacccagagc (SEQ ID NO: 131) CX0702gtgcagccaccgtacgcttaatctccactttggtg (SEQ ID NO: 132) CX0325tgcttgctcaactctacgtc (SEQ ID NO: 133) CX0289gctttcaccgcaggtacttccgtagctggccagtctggcc (SEQ ID NO: 134) CX0687cgctccatgggccaccttggccgctgccaccgctcgagcc (SEQ ID NO: 135) CX0700cacttgtcacgaattcggaggtccagctggtagaaag (SEQ ID NO: 136) CX0701ggcccttggtgctagcgctcgacactgtaaccagagtac (SEQ ID NO: 137)

TABLE 27 Sequences for heavy and light chain anti-VEGF antibodypFIL2-CL-hk anti-VEGF Lc (pFIL2-VEGF-Lc)gatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 138)DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQG LSSPVTKSFNRGEC (SEQID NO: 139)

As described above, the mask 306, used for anti-VEGF AA development didnot efficiently mask the target binding over long exposure to target,due to low affinity of the MM for the AB. One approach to increasing theaffinity of the MM is to subject the peptide to affinity maturation asdescribed below.

Library Construction for Affinity Maturation

The 306 anti-VEGF MM was affinity matured by using a soft randomizationapproach. An ecpX cell display library was constructed in with thenucleotide ratios shown in Table 28. The final library diversity (306SR) was approximately 2.45×10⁸.

TABLE 28 Original Base Ratio of Bases G G = 70%; T = 8%; A = 11%; C =11% T T = 70%; G = 8%; A = 11%; C = 11% A A = 80%; G = 5%; T = 6%; C =9% C C = 80%; G = 5%; T = 6%; A = 9%306 SR Library Screening

An initial MACS round was performed with protein-A labeled magneticbeads and a number of cells that provided greater than 100× oversamplingof the library. Prior to magnetic selection the cells were incubatedwith 100 nM anti-VEGF IgG and 10 μM 306 peptide (306P, PCSEWQSMVQPRCYYG(SEQ ID NO: 140)), to reduce the binding of variants with equal or loweraffinity than the original 306 sequence. Magnetic selection resulted inthe isolation of 2×10⁷ cells.

The first round of FACS sorting was performed on cells labeled with 1 nMDyLight (fluor 530 nM)-anti-VEGF. To apply selective pressure to thepopulation, the second and third round of FACS was performed on cellslabeled with 1 nM DyLight-anti-VEGF in the presence of 100 nM 306P.Selection gates were set so that only 5% of cells with the strongestbinding were collected. The population of cells sorted in the thirdround were first incubated with 10 nM DyLight-anti-VEGF followed byaddition of 306P to a final concentration of 100 nM and incubated at 37°C. for 20 minutes. The brightest 2% of the positive population wascollected, representing binding that was not competed by 306P. FACSrounds 5 through 7 were done as follows; the populations were labeledwith 10 nM DyLight labeled anti-VEGF and then competed off withunlabeled VEGF (100 nM) at 37° C. for 7, 10, and 15 minutes,respectively. The brightest 1% were sorted in FACS rounds 5 through 7.

TABLE 29 306SR M1F7 peptide sequences JS306 PCSEWQSMVQPRCYYG (SEQ ID NO:141) JS1825 SCTAWQSMVEQRCYFG (SEQ ID NO: 142) 3X JS1826 PCSKWESMVEQRCYFA(SEQ ID NO: 143) JS1827 PCSAWQSMVEQRCYFG (SEQ ID NO: 144) 2X JS1829PCSKWESMVLQSCYFG (SEQ ID NO: 145) 4X JS1830 TCSAWQSMVEQRCYFG (SEQ ID NO:146) 2X JS1837 TCSQWESMVEPRCYFG (SEQ ID NO: 147)306SR Affinity Matured Peptide Analysis

Binding of the eCPX3.0 clones 306, JS1825, JS1827, and JS1829 wereanalyzed on FACS at 3 different concentrations of DyLight labeledanti-VEGF. The binding curves are shown in FIG. 21. All three of theaffinity matured peptides displayed at least 10 fold higher affinitythan 306P.

Construction of Anti-VEGF AAs

Affinity matured ecpX3.0 clones (JS1825, JS1827, and JS1829) were PCRamplified using primers CX0289 and CX0687 and cloned intopFIL2-306mVEGF-Lc using the SfiI restriction sites to produce thevectors pFIL2-1825mVEGF-Lc, pFIL2-1827mVEGF-Lc, and pFIL2-1829mVEGF-Lc.The nucleotide and amino acid sequences are provided in the tablesfollowing. Parentheses delineate the demarcations between the varioussequence domains: (Linker)(MM)(Linker)(CM)(Linker)(AB).

TABLE 30 Sequences of anti-VEGF AA: pFIL2-CL-hk anti-VEGF mmp-9 306 Lc(pFIL2- 306mVEGF-Lc)ggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 148)Linker         MM         Linker      CM     Linker     AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGG)(QVHMPLGFLGP)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 149)

TABLE 31 Sequences of anti-VEGF AA: pFIL2-CL-hk anti-VEGF mmp-9 1825 Lc(pFIL2- 1825mVEGF-Lc)ggccagtctggccagtcgtgtacggcgtggcagtcgatggtggagcagcgttgctattttgggggctcgagcggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 150)Linker         MM         Linker      CM     Linker     AB(GQSGQ)(SCTAWQSMVEQRCYFG)(GSSGGSGQGGQ)(VHMPLGFLGP)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 151)

TABLE 32 Sequences of anti-VEGF AA: pFIL2-CL-hk anti-VEGF mmp-9 1827 Lc(pFIL2- 1827mVEGF-Lc)ggccagtctggccagccgtgttctgcgtggcagtctatggtggagcagcgttgctattttgggggctcgagcggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 152) Linker       MM          Linker      CM     Linker     AB(GQSGQ)(PCSAWQSMVEQRCYFG)(GSSGGSGQGG)(QVHMPLGFLGP)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 153)

TABLE 33 Sequences of anti-VEGF AA: pFIL-CL-hk anti-VEGFmmp-9 1829 Lc (pFIL2-1829mVEGF-Lc)ggccagtctggccagccgtgttctaagtgggaatcgatggtgctgcagagttgctattttggcggctcgagcggtggcagcggccaaggtggccaagtgcacatgccactgggcttcctgggtccgggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttageggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 154)Linker MM Linker CM Linker AB(GQSGQ)(PCSKWESMVLQSCYFG)(GSSGGSGQGG)(QVHMPLGFLGP)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 155)Expression and Purification of Anti-VEGF Antibody and AA

3 μg of pFIL-VEGF-Hc and 3 μg pFIL2-VEGF-Lc were co-transfected intoCHO—S cells (Invitrogen) using Lipofectamine 200 (Invitrogen) accordingto manufacturers protocol. Transfected cells were cultured in FreestyleCHO media (Invitrogen) and selected for resistance to zeocin andblasticidin. Individual clones were isolated by limiting dilution andselected for expression of human IgG capable of binding EGFR by ELISA.All antibodies and AAs are purified by Protein-A chromatography usingstandard techniques.

Likewise, 3 μg of each expression vector for AA light chainspFIL2-306mVEGF-Lc, pFIL2-1825mVEGF-Lc, pFIL2-1827mVEGF-Lc, orpFIL2-1829mVEGF-Lc was co-transfected into CHO—S cells with 3 μgpFIL-VEGF-Hc. Transfected cells were cultured in Freestyle CHO media(Invitrogen) and selected for resistance to zeiocin and blasticiidin.Inkdivideual clones were isolated by limiting dilution and selected forexpression of human IgG capable of binding EGFR by ELISA.

Target Displacement Assay of Anti-VEGF Antibody and AA

VEGF is adsorbed to the wells of a 96 well micro-titer plate, washed andblocked with milk protein. About 25 ml of culture media containinganti-VEGF antibody or anti-VEGF AA's containing the MM's JS306, JS1825,JS1827 and JS 1829 is added to the coated wells and incubated for about1, 2, 4, 8 or 24 hours. Following incubation the wells are washed andthe extent of bound AA's measured by anti-huIgG immunodetection.

Example 16: Construction of an Anti-EGFR IgG AA

Construction of an Anti-EGFR IgG Antibody

The C225 light chain variable region gene was synthesized by assemblyPCR using oligos CX638-CX655 as in Bessette et al., Methods in MolecularBiology, vol. 231. The resulting product was digested with BamHI/NotIand ligated to the large fragment of pXMal digested with BamHI/NotI tocreate plasmid pX-scFv225-Vk. Similarly, the C225 heavy chain variableregion gene was synthesized by assembly PCR using oligos CX656-CX677,digested with BglII/NotI and ligated to pXMal BamHI/NotI to createplasmid pX-scFv225-Vh. The variable light chain gene was then clonedfrom pX-scFv225-Vk as a BamHI/NotI fragment into the pX-scFv225-Vhplasmid at BamHI/Not to create the plasmid pX-scFv225m-HL, containingthe scFv gene based on C225.

The IL2 signal sequence was moved from pINFUSE-hIgG1-Fc2 (InvivoGen) asa KasI/NcoI fragment to pFUSE2-CLIg-hk (InvivoGen) digested withKasI/NcoI, resulting in plasmid pFIL2-CL-hk. The IL2 signal sequence wasalso moved from pINFUSE-hIgG1-Fc2 as a KasI/EcoRI fragment topFUSE-CHIg-hG1 (InvivoGen) digested with KasI/EcoRI (large and mediumfragments) in a three-way ligation, resulting in plasmid pFIL-CHIg-hG1.

The human IgG light chain constant region was site specifically mutatedby amplification from plasmid pFIL2-CL-hk with oligos CX325/CX688,digestion with BsiWI/NheI, and cloning into pFIL2-CL-hk at BsiWI/NheI,resulting in plasmid pFIL2-CL₂₂₅.

The human IgG heavy chain constant region was site specifically mutatedby amplification from plasmid pFIL-CHIg-hG1 in three segments witholigos CX325/CX689, CX690/CX692, and CX693/CX694, followed by overlapPCR of all three products using outside primers CX325/CX694. Theresulting product was digested with EroRI/AvrII and cloned intopINFUSE-hIgG1-Fc2 at EcoRI/NheI, resulting in plasmid pFIL-CH₂₂₅.

The variable light chain gene segment was amplified from pX-scFv225m-HLwith oligos CX695/CX696, digested with BsaI, and cloned into pFIL2-CL₂₂₅at EcoRI/BsiWI, resulting in the C225 light chain expression vectorpFIL2-C225-light.

The variable heavy chain gene segment was amplified from pX-scFv225m-HLwith oligos CX697/CX698, digested with BsaI, and cloned into pFIL-CH₂₂₅at EcoRI/NheI, resulting in the C225 heavy chain expression vectorpFIL-C225-heavy.

TABLE 33 Primers Used in the Construction of anti-EGFR IgG antibodyCX268 ccgcaggtacctcgagcgctagccagtctggccag (SEQ ID NO: 156) CX325tgcttgctcaactctacgtc (SEQ ID NO: 157) CX370 aacttgtttattgcagctt (SEQ IDNO: 158) CX448 gagttttgtcggatccaccagagccaccgctgccaccgctcgagcc (SEQ IDNO: 159) CX638 gcgtatgcaggatccggcggcgatattctgctgacccaga (SEQ ID NO: 160)CX639 cacgctcagaatcaccgggctctgggtcagcagaatatcg (SEQ ID NO: 161) CX640gcccggtgattctgagcgtgagcccgggcgaacgtgtgag (SEQ ID NO: 162) CX641ggctcgcgcggcagctaaagctcacacgttcgcccgggct (SEQ ID NO: 163) CX642ctttagctgccgcgcgagccagagcattggcaccaacatt (SEQ ID NO: 164) CX643gtgcgctgctgataccaatgaatgttggtgccaatgctct (SEQ ID NO: 165) CX644cattggtatcagcagcgcaccaacggcagcccgcgcctgc (SEQ ID NO: 166) CX645ttcgctcgcatatttaatcagcaggcgcgggctgccgttg (SEQ ID NO: 167) CX646tgattaaatatgcgagcgaaagcattagcggcattccgag (SEQ ID NO: 168) CX647tgccgctgccgctaaagcggctcggaatgccgctaatgct (SEQ ID NO: 169) CX648ccgctttagcggcagcggcagcggcaccgattttaccctg (SEQ ID NO: 170) CX649ctttccacgctgttaatgctcagggtaaaatcggtgccgc (SEQ ID NO: 171) CX650agcattaacagcgtggaaagcgaagatattgcggattatt (SEQ ID NO: 172) CX651gttgttgttctgctggcaataataatccgcaatatcttcg (SEQ ID NO: 173) CX652attgccagcagaacaacaactggccgaccacctttggcgc (SEQ ID NO: 174) CX653tcagttccagtttggtgcccgcgccaaaggtggtcggcca (SEQ ID NO: 175) CX654gggcaccaaactggaactgaaacgcggccgccatcaccat (SEQ ID NO: 176) CX655ctcccacgcgtatggtgatgatggtgatggcggccgcgtt (SEQ ID NO: 177) CX656cgtatgcaagatctggtagcggtacccaggtgcagctgaa (SEQ ID NO: 178) CX657ccaggcccgggccgctctgtttcagctgcacctgggtacc (SEQ ID NO: 179) CX658acagagcggcccgggcctggtgcagccgagccagagcctg (SEQ ID NO: 180) CX659ctcacggtgcaggtaatgctcaggctctggctcggctgca (SEQ ID NO: 181) CX660agcattacctgcaccgtgagcggctttagcctgaccaact (SEQ ID NO: 182) CX661gcgcacccaatgcacgccatagttggtcaggctaaagccg (SEQ ID NO: 183) CX662atggcgtgcattgggtgcgccagagcccgggcaaaggcct (SEQ ID NO: 184) CX663aaatcacgcccagccattccaggcctttgcccgggctctg (SEQ ID NO: 185) CX664ggaatggctgggcgtgatttggagcggcggcaacaccgat (SEQ ID NO: 186) CX665ctggtaaacggggtgttataatcggtgttgccgccgctcc (SEQ ID NO: 187) CX666tataacaccccgtttaccagccgcctgagcattaacaaag (SEQ ID NO: 188) CX667cacctggcttttgctgttatctttgttaatgctcaggcgg (SEQ ID NO: 189) CX668ataacagcaaaagccaggtgttttttaaaatgaacagcct (SEQ ID NO: 190) CX669tcgcggtatcgttgctttgcaggctgttcattttaaaaaa (SEQ ID NO: 191) CX670gcaaagcaacgataccgcgatttattattgcgcgcgcgcg (SEQ ID NO: 192) CX671tcataatcataataggtcagcgcgcgcgcgcaataataaa (SEQ ID NO: 193) CX672ctgacctattatgattatgaatttgcgtattggggccagg (SEQ ID NO: 194) CX673gctcacggtcaccagggtgccctggccccaatacgcaaat (SEQ ID NO: 195) CX674gcaccctggtgaccgtgagcgcgggtggtagcggtagcgg (SEQ ID NO: 196) CX675taccgccgcctccagatcctccgctaccgctaccacccgc (SEQ ID NO: 197) CX676aggatctggaggcggcggtagtagtggtggaggatccggt (SEQ ID NO: 198) CX677tggtgatggcggccgcggccaccggatcctccaccactac (SEQ ID NO: 199) CX688cgagctagctccctctacgctcccctgttgaagctctttg (SEQ ID NO: 200) CX690acaagcgcgttgagcccaaatcttgtg (SEQ ID NO: 201) CX692cagttcatcccgggatgggggcagggtg (SEQ ID NO: 202) CX693ccccatcccgggatgaactgaccaagaaccaggtcagc (SEQ ID NO: 203) CX694ctggccacctaggactcatttaccc (SEQ ID NO: 204) CX695gcactggtctcgaattcggatattctgctgacccagag (SEQ ID NO: 205) CX696ggtgcggtctccgtacgtttcagttccagtttggtg (SEQ ID NO: 206) CX697gcactggtctcgaattcgcaggtgcagctgaaacagag (SEQ ID NO: 207) CX698gagacggtctcgctagccgcgctcacggtcaccag (SEQ ID NO: 208) CX730tgcgtatgcaagatctggtagcggtaccgatattctgctgacccagag (SEQ ID NO: 209) CX731actactaccgccgcctccagatcctccgctaccgctaccacctttcagttccagtttggtg (SEQ IDNO: 210) CX732tctggaggcggcggtagtagtggtggaggctcaggcggccaggtgcagctgaaacagag (SEQ ID NO:211) CX733 gatggtgatggcggccgcgcgcgctcacggtcaccag (SEQ ID NO: 212) CX735tgtcggatccaccgctaccgcccgcgctcacggtcaccag (SEQ ID NO: 213) CX740tcacgaattcgcaaggccagtctggccagggctcgagcggtggcagcggtggctctggtggatccggcggtggca(SEQ ID NO: 214) CX741tggtggatccggcggtggcagcggtggtggctccggcggtaccggcggtagcggtagatctgacaaaactcacac(SEQ ID NO: 215) CX747 gatccccgtctccgccagtcaaaatgatgccggaaggcggtac (SEQID NO: 216) CX748 cgccttccggcatcattttgactggcggagacggg (SEQ ID NO: 217)Construction of Expression Vectors for Anti-EGFR AAs

Plasmid pX-scFv225m-HL was PCR amplified in separate reactions withprimers CX730/CX731 and CX732/CX733, and the resulting products wereamplified by overlap PCR with outside primers CX730/CX733, digested withBglII/NotI, and cloned into pXMal at BamHI/NotI, resulting in plasmidpX-scFv225m-LH.

Linker sequence was added to the N-terminal side of the human IgG Fcfragment gene by PCR amplification of pFUSE-hIgG-Fc2 in a reaction withoverlapping forward primers CX740, CX741 and reverse primer CX370. Theresulting product was digested with EcoRI/BglII, and the ˜115 bpfragment was cloned into pFUSE-hIgG-Fc2 at EcoRI/BglII. The resultingplasmid was digested with KpnI/BglII, and the large fragment was ligatedto the KpnI/BamHI-digested PCR product of amplifying pX-scFv225m-LH witholigos CX736/CX735, resulting in plasmid pPHB3734.

The resulting plasmid was digested with SfiI/XhoI, and masking peptide3690 was cloned in as an SfiI/XhoI fragment of pPHB3690, resulting inplasmid pPHB3783.

The protease substrate SM984 was added by digesting the resultingplasmid with BamHI/KpnI and ligating the product of annealing thephosphorylated oligos CX747/CX748, resulting in plasmid pPHB3822.

The tandem peptide mask was constructed by digesting the resultingplasmid with XhoI, dephosphorylating the 5′ ends, and cloning in theXhoI-digested PCR product of amplifying pPHB3579 with primersCX268/CX448, resulting in plasmid pPHB3889.

The masking region, linker, substrate, and light chain variable regionof pPHB3783, pPHB3822, and pPHB3889 were amplified by PCR with primersCX325/CX696, digested with EcoRI/BsiWI, and cloned into pFIL2-CL₂₂₅ atEcoRI/BsiWI, resulting in the AA light chain expression vectorspPHB4007, pPHB3902, and pPHB3913 respectively.

Affinity matured masking peptides were swapped into the AA light chainexpression vectors by cloning as SfiI/XhoI fragments. Proteasesubstrates were swapped in as BamHI/KpnI compatible fragments.

Expression and Purification of the Anti-EGFR Antibody and AAs

3 μg of pFIL-CH₂₂₅-HL and 3 μg pFIL2-CH₂₂₅-light were co-transfectedinto CHO—S cells (Invitrogen) using Lipofectamine 200 (Invitrogen)according to manufacturers protocol. Transfected cells were cultured inFreestyle CHO media (Invitrogen) and selected for resistance to zeocinand blasticidin. Individual clones were isolated by limiting dilutionand selected for expression of human IgG capable of binding EGFR byELISA. All antibodies and AAs are purified by Protein-A chromatographyusing standard techniques.

Likewise, 3 μg of each expression vector for AA light chains wasco-transfected into CHO—S cells with 3 μg pFIL-CH₂₂₅-HL. Transfectedcells were cultured in Freestyle CHO media (Invitrogen) and selected forresistance to zeiocin and blasticiidin. Inkdivideual clones wereisolated by limiting dilution and selected for expression of human IgGcapable of binding EGFR by ELISA.

Screening of the Affinity Matured Anti-EGFR MM Library.

An initial MACS round was performed with SA dynabeads and 1.4×10⁸ cellsfrom the ecpX3-755 library. Prior to magnetic selection the cells wereincubated with 3 nM biotin labeled C225Mab. Magnetic selection resultedin the isolation of 6×10⁶ cells. The first round of FACS sorting wasperformed on 2×10⁷ cells labeled with 0.1 nM DyLight (fluor 530nM)-C225Mab and resulted in isolation of 1.5×10⁵ cells with positivebinding. To apply increased selective pressure to the population, thesecond round of FACS was performed on cells labeled with 10 nMDyLight-C225Mab in the presence of 100 μM 3690 peptide (CISPRGC (SEQ IDNO: 1)) at 37° C. To further increase the selction pressure the 3^(rd)and 4^(th) rounds were performed on cells labeled with 100 nMDyLight-C225Fab in the presence of 100 μM3690 peptide (CISPRGC (SEQ IDNO: 1)) at 37° C. The brightest 1% of the positive population werecollected, representing binding that was not competed by 3690 peptide.On cell affinity measurements of individual clones isolated from theabove screen revealed three peptides, 3954(CISPRGCPDGPYVM (SEQ ID NO:218)), 3957(CISPRGCEPGTYVPT (SEQ ID NO: 219)) and 3958(CISPRGCPGQIWHPP(SEQ ID NO: 220)) with affinities for C225 at least 100 fold greaterthan 3690 (CISPRGC (SEQ ID NO: 1)). These three MMs were incorporatedinto anti-EGFR AAs. FIG. 22 shows the process for affinity maturation ofsome of the EGFR MM's.

Affinity Measurement for C225 MMs

On-cell affinity measurement of C225 Fab binding to MM's 3690, 3954 and3957. Binding of the eCPX3.0 clones 3690, 3954 and 3957 were analyzed onFACS at 3 different concentrations of DyLight labeled anti-EGFR Fab. Thebinding curves are shown in FIG. 23. MMs 3954 and 3957 displayed atleast 100 fold higher affinity than 3690.

Target Displacement Assay for Anti-EGFR AAs

EGFR was adsorbed to the wells of a 96 well micro-titer plate, washedand blocked with milk protein. 25 ml of culture media containing 2 nManti-EGFR antibody or anti-EGFR AA's containing the MM's 3690, 3957,3954 and 3960/3579 was added to the coated wells and incubated for 1, 2,4, 8 or 24 hours. Following incubation the wells were washed and theextent of bound AA's measured by anti-huIgG immunodetection. Anti-EGFRAA binding was normalized to anti-EGFR antibody binding (100%) fordirect comparison of the masking efficiency in the AA context. Theextents of equilibrium binding as a percent of parental or unmodifiedantibody binding are shown in Table 34 and FIG. 24. Whereas MMs 3954 and3957 display the same affinity, 100 times higher than 3609, 3954 is atleast 2 times more efficient at inhibiting target binding. The sequencesof the C225 heavy and light chains, MMs, and AAs are provided in thetables following. Nucleotide and amino acid sequences provided in thetables following. Parentheses delineate the demarcations between thevarious sequence domains: (Linker)(MM)(Linker)(CM)(Linker)(AB).

TABLE 34 C225 TDA: Percent of parental antibody binding ± SEM at eachtime point Time (hours) 3690 AA 3954 AA 3975 AA 3690/3579 AA 1 15.5 ±4.2 4.4 ± 1.8 7.3 ± 2.0 3.6 ± 1.2 2 19.3 ± 6.0 6.0 ± 2.0 9.3 ± 2.8 2.1 ±0.6 4 21.5 ± 5.0 7.6 ± 1.7 12.8 ± 2.3  3.3 ± 1.2 8 27.6 ± 7.4 9.7 ± 0.414.9 ± 0.03 3.0 ± 1.6 24 20.0 ± 9.1 13.4 ± 1.2  22.3 ± 2.6  2.8 ± 0.1

TABLE 35 C225 Heavy Chaincaggtgcagctgaaacagagcggcccgggcctggtgcagccgagccagagcctgagcattacctgcaccgtgagcggctttagcctgaccaactatggcgtgcattgggtgcgccagagcccgggcaaaggcctggaatggctgggcgtgatttggagcggcggcaacaccgattataacaccccgtttaccagccgcctgagcattaacaaagataacagcaaaagccaggtgttttttaaaatgaacagcctgcaaagcaacgataccgcgatttattattgcgcgcgcgcgctgacctattatgattatgaatttgcgtattggggccagggcaccctggtgaccgtgagcgcggctagcaccaagggcccatcggtcttccccctggcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtcctacagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagcgcgttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagaccctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgtgtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgaactgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctctacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctctccctgtctccgggtaaa (SEQ ID NO: 221)QVQLKQSGPGLVQPSQSLSITCTVSGFSLTNYGVHWVRQSPGKGLEWLGVIWSGGNTDYNTPFTSRLSINKDNSKSQVFFKMNSLQSNDTAIYYCARALTYYDYEFAYWGQGTLVTVSAASTKGPSVFPLAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKRVEPKSCDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPGK (SEQ ID NO: 222)

TABLE 36 Sequence of 3690-SM984-C225 Light ChainCaaggccagtctggccagtgcatctcgccccgtggttgtggaggctcgagcggtggcagcggtggctctggtggatccccgtctccgccagtcaaaatgatgccggaaggcggtacccagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagcg (SEQ ID NO: 223)Linker    MM         Linker      CM    Linker       AB(QGQSGQ)(CISPRGC)(GGSSGGSGGSGGS)(PSPPVKMMPE)(GG)(TQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA) (SEQ ID NO: 224)

TABLE 37 Sequence of 3579-NSUB-C225 Light Chaincaaggccagtctggccagggttcacattgtctcattcctattaacatgggcgcgccgtcatgcggctcgagcggtggcagcggtggctctggtggatccggcggtggcagcggtggtggctccggcggtacccagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagcg (SEQ ID NO: 225)Linker       MM                 Linker                AB(QGQSGQ)(GSHCLIPINMGAPSC)(GSSGGSGGSGGSGGGSGGGSGG)(TQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA) (SEQ ID NO: 226)

TABLE 38 Sequence of 3690-3579-SM984-C225 Light Chaincaaggccagtctggccagtgcatctcgccccgtggttgtggaggctcgagcgctagccagtctggccagggttcacattgtctcattcctattaacatgggcgcgccgtcatgcggctcgagcggtggcagcggtggctctggtggatccccgtctccgccagtcaaaatgatgccggaaggcggtacccagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagcg (SEQ ID NO: 227) Linker              MM                   Linker       CM(QGQSGQ)(CISPRGCGGSSASQSGQGSHCLIPINMGAPSC)(GSSGGSGGSGGS)(PSPPVKM MPE)Linker     AB(GG)(TQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA) (SEQ ID NO: 228)

TABLE 39 Sequence of 3954-NSUB-C225 Light Chaincaaggccagtctggccagtgcatctcacctcgtggttgtccggacggcccatacgtcatgtacggctcgagcggtggcagcggtggctctggtggatccggcggtggcagcggtggtggctccggcggtacccagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagcg (SEQ ID NO: 229)Linker        MM                Linker               AB(QGQSGQ)(CISPRGCPDGPYVMY)(GSSGGSGGSGGSGGGSGGGSGG)(TQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA) (SEQ ID NO: 230)

TABLE 40 Sequence of 3957-NSUB-C225 Light Chaincaaggccagtctggccagtgcatctcacctcgtggttgtgagcctggcacctatgttccaacaggctcgagcggtggcagcggtggctctggtggatccggcggtggcagcggtggtggctccggcggtacccagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagcg (SEQ ID NO: 231)Linker        MM                Linker                AB(QGQSGQ)(CISPRGCEPGTYVPT)(GSSGGSGGSGGSGGGSGGGSGG)(TQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA) (SEQ ID NO: 232)

TABLE 41 Sequence of 3958-NSUB-C225 Light Chaincaaggccagtctggccagtgcatctcacctcgtggttgtccgggccaaatttggcatccacctggctcgagcggtggcagcggtggctctggtggatccggcggtggcagcggtggtggctccggcggtacccagatcttgctgacccagagcccggtgattctgagcgtgagcccgggcgaacgtgtgagctttagctgccgcgcgagccagagcattggcaccaacattcattggtatcagcagcgcaccaacggcagcccgcgcctgctgattaaatatgcgagcgaaagcattagcggcattccgagccgctttagcggcagcggcagcggcaccgattttaccctgagcattaacagcgtggaaagcgaagatattgcggattattattgccagcagaacaacaactggccgaccacctttggcgcgggcaccaaactggaactgaaacgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagcg (SEQ ID NO: 233)Linker        MM                Linker                AB(QGQSGQ)(CISPRGCPGQIWHPP)(GSSGGSGGSGGSGGGSGGGSGG)(TQILLTQSPVILSVSPGERVSFSCRASQSIGTNIHWYQQRTNGSPRLLIKYASESISGIPSRFSGSGSGTDFTLSINSVESEDIADYYCQQNNNWPTTFGAGTKLELKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGA)(SEQ ID NO: 234)

TABLE 42 Sequences of C225 MMs: 3690 CISPRGC (SEQ ID NO: 1) 3579GSHCLIPINMGAPSC (SEQ ID NO: 236) 3690-3579CISPRGCGGSSASQSGQGSHCLIPINMGAPSC (SEQ ID NO: 237) 3954CISPRGCPDGPYVMY (SEQ ID NO: 238) 3957 CISPRGCEPGTYVPT (SEQ ID NO: 239)3958 CISPRGCPGQIWHPP (SEQ ID NO: 240) 4124CNHHYFYTCGCISPRGCPG (SEQ ID NO: 241) 4125ADHVFWGSYGCISPRGCPG (SEQ ID NO: 242) 4127CHHVYWGHCGCISPRGCPG (SEQ ID NO: 243) 4133CPHFTTTSCGCISPRGCPG (SEQ ID NO: 244) 4137CNHHYHYYCGCISPRGCPG (SEQ ID NO: 245) 4138CPHVSFGSCGCISPRGCPG (SEQ ID NO: 246) 4140CPYYTLSYCGCISPRGCPG (SEQ ID NO: 247) 4141CNHVYFGTCGCISPRGCPG (SEQ ID NO: 248) 4143CNHFTLTTCGCISPRGCPG (SEQ ID NO: 249) 4148CHHFTLTTCGCISPRGCPG (SEQ ID NO: 250) 4157YNPCATPMCCISPRGCPG (SEQ ID NO: 251)EGFR MM Consensus Sequences

The consensus sequences for the EGFR MMs are provided below. The 3690 MMconsensus (CISPRGC (SEQ ID NO: 1)) is one major consensus sequence.

TABLE 43 C225 EGFR MM Consensus Sequences PHB4124CNHHYFYTCGCISPRGCG (SEQ ID NO: 252) PHB4137CNHHYHYYCGCISPRGCG (SEQ ID NO: 253) PHB4141CNHVYFGTCGCISPRGCG (SEQ ID NO: 254) PHB4127CHHVYWGHCGCISPRGCG (SEQ ID NO: 255) PHB4133CPHFTTTSCGCISPRGCG (SEQ ID NO: 256) PHB4143CNHFTLTTCGCISPRGCG (SEQ ID NO: 257) PHB4148CHHFTLTTCGCISPRGCG (SEQ ID NO: 258) PHB4140CPYYTLSYCGCISPRGCG (SEQ ID NO: 259) PHB4138CPHVSFGSCGCISPRGCG (SEQ ID NO: 260) PHB4125ADHVFWGSYGCISPRGCG (SEQ ID NO: 261) PHB4157YNPCATPMCCISPRGCG (SEQ ID NO: 262) PHB4127CHHVYWGHCGCISPRGCG (SEQ ID NO: 263)EGFR consensus Sequences from the 2^(nd) round ofscreening for higher affinity masksC(N/P)H(H/V/F)(Y/T)(F/W/T/L)(Y/G/T/S)(T/S/Y/H) CGCISPRGCG (SEQ ID NO: 2)CISPRGCGQPIPSVK (SEQ ID NO: 264) CISPRGCTQPYHVSR (SEQ ID NO: 265)CISPRGCNAVSGLGS (SEQ ID NO: 266)

Example 17: Selective Substrate/CM Discovery and Testing

The section below the process for selective substrate discovery andtesting for a number of exemplary enzymes.

uPA selective substrate discovery

uPA-selective substrates were isolated from an 8eCLiPS bacterial libraryconsisting of ˜10⁸ random 8-mer substrates expressed as N-terminalfusions on the surface of E. coli. Alternating rounds of positive andnegative selections by FACS were used to enrich for substrates optimizedfor cleavage by uPA and resistant to cleavage by the off-target serineproteases klk5 and 7. The naive library was incubated with 8 μg/ml uPAfor 1 h at 37° C. followed by labeling with SAPE(red) and yPET mona(green). Cleavage by uPA results in loss of the SAPE tag and allows forsorting of bacteria expressing uPA substrates (green only, positiveselection) from bacteria expressing uncleaved peptides (red+green). uPAsubstrates were sorted by FACS and the enriched pool was amplified andthen incubated with 5 ng/ml KLK5 and 7 for 1 h at 37° C., labeled withSAPE and yPET mona, and sorted for lack of cleavage by these off-targetproteases (red+green, negative selection). The pool was amplified andsorted with 4 additional alternating rounds of positive and negativeFACS using decreasing concentrations of uPA (4 μg/ml, 2 μg/ml) andincreasing concentrations of klk5 and 7 (5 ng/ml, 10 ng/ml). Individualclones from the last 3 rounds of FACS were sequenced and grouped intoseveral consensuses (Table 44). Clones from each consensus were thenanalyzed individually for cleavage by a range of concentrations of uPA,klk5 and 7 and plasmin for specificity of cleavage by on versusoff-target proteases in Table 44. FIG. 25 shows that unlike the uPAcontrol and substrate SM16, KK1203, 1204 and 1214 show resistance tocleavage by KLK5, KLK7 and Plasmin.

TABLE 44 uPA Consensus sequences (SEQ ID NOS 267-280, respectively, inorder or appearance)   kk1203 (1) (1)

kk1206 (1)

kk1216 (1)

Consensus (1) TARGPS W   kk1204 (1) (1)

kk1208 (1)

kk1211 (1)

kk1214 (1)

Consensus (1)   LTGRSGA   kk1217 (1) (1)

kk1219 (1)

kk1196 (1)

kk1201 (1)

Consensus (1)   RGPAPlasmin Selective Substrate Discovery

Plasmin-selective substrates were isolated from a second generationplasmin 10eCLiPS bacterial library consisting of ˜10⁸ random 10-mersubstrates expressed as N-terminal fusions on the surface of E. coli(ref). Alternating rounds of positive and negative selections by FACSwere used to enrich for substrates optimized for cleavage by plasmin andresistant to cleavage by the off-target matrix metalloproteinases(represented by MMP-9) and serine proteases (represented by klk5 andklk7).

The second generation plasmin 10eCLiPS library was based on a consensussequence identified in-house by selecting the naive 8eCLiPS for rapidlycleaved plasmin substrates using concentrations as low as 30 μM plasminfor selection. Individual residues within the 10mer were either random(n=20), restricted (l<n>20) or fixed (n=1) to bias the peptide towardthe consensus sequence while allowing flexibility to down-select awayfrom unfavorable off-target sequences.

The second generation plasmin 10eCLiPS library was incubated with 300 pMplasmin for 1 h at 37° C. followed by labeling with SAPE(red) and yPETmona (green). Cleavage by plasmin results in loss of the SAPE tag andallows for sorting of bacteria expressing plasmin substrates (greenonly, positive selection) from bacteria expressing uncleaved peptides(red+green). plasmin substrates were sorted by FACS and the enrichedpool was amplified and then incubated with 80 U/ml MMP-9 2 h at 37° C.,labeled with SAPE and yPET mona, and sorted for lack of cleavage bythese off-target proteases (red+green, negative selection). The pool wasamplified and sorted with 4 additional alternating rounds of positiveand negative FACS using plasmin (round three at 100 pM or 300 pM, roundfive at 100 pM or 300 pM) and klk5 and 7 (Round four at 100 ng/ml, roundsix at 200 ng/ml). Individual clones from each the last 2 rounds of FACSwere sequenced (Table 45). Clones from each consensus were then analyzedindividually for cleavage by plasmin, MMP-9, klk5 and klk7 forspecificity of cleavage by on versus off-target proteases.Representative data showing increased specificity towards Plasmincleavage is shown in FIG. 26. FIG. 26 shows that unlike a non-optimizedsubstrate, the optimized substrates Plas1237, Plas129 and Plas 1254 showresistance to cleavage by KLK5, KLK7.

TABLE 45 Peptide sequences derived from three rounds of Positiveselection for Plasmin cleavage and negative selection for MMP9, KLK5 andKLK7 SM1191 EHPRVKVVSE (SEQ ID NO: 281) SM1197 PPPDMKLFPG (SEQ ID NO:282) SM1200 PPPVLKLLEW (SEQ ID NO: 283) SM1203 VLPELRSVFS (SEQ ID NO:284) SM1206 APPSFKLVNA (SEQ ID NO: 285) SM1212 PPPEVRSFSV (SEQ ID NO:286) SM1214 ALPSVKMVSE (SEQ ID NO: 287) SM1215 ETPSVKTMGR (SEQ ID NO:288) SM1219 AIPRVRLFDV (SEQ ID NO: 289) SM1224 GLGTPRGLFA (SEQ ID NO:290) SM1276 DRPKVKTMDF (SEQ ID NO: 291) SM1275 RVPKVKVMLD (SEQ ID NO:292) SM1274 APPLVKSMVV (SEQ ID NO: 293) SM1272 REPFMKSLPW (SEQ ID NO:294) SM1270 PVPRLKLIKD (SEQ ID NO: 295) SM1269 KGPKVKVVTL (SEQ ID NO:296) SM1268 ERPGVKSLVL (SEQ ID NO: 297) SM1267 NZPRVRLVLP (SEQ ID NO:298) SM1265 PRPFVKSVDQ (SEQ ID NO: 299) SM1263 RFPSLKSFPL (SEQ ID NO:300) SM1261 ESPVMKSMAL (SEQ ID NO: 301) SM1260 VAPQLKSLVP (SEQ ID NO:302) SM1255 APPLVKSMVV (SEQ ID NO: 303) SM1254 NMPSFKLVTG (SEQ ID NO:304) SM1245 DRPEMKSLSG (SEQ ID NO: 305) SM1244 EQPEVKMVKG (SEQ ID NO:306) SM1243 AVPKVRVVPE (SEQ ID NO: 307) SM1241 DLPLVKSLPS (SEQ ID NO:308) SM1240 EAPKVKALPK (SEQ ID NO: 309) SM1239 GFPHMKTFQH (SEQ ID NO:310) SM1238 YDPZVKVVLA (SEQ ID NO: 311) SM1237 ASPTMKTVGL (SEQ ID NO:312) SM1236 DVPPMKTLRP (SEQ ID NO: 313) SM1235 AFPDMRSVRS (SEQ ID NO:314) SM1234 SAPYFRMMDM (SEQ ID NO: 315) SM1233 EKPRMKLFQG (SEQ ID NO:316) SM1231 YVPRVKALEM (SEQ ID NO: 317)uPA Enzyme Activated AA Sequences

Nucleotide and amino acid sequences of uPA enzyme-activated anti VEGFlight chain AAs are provided in the tables below. Parentheses delineatethe demarcations between the various sequence domains:(Linker)(MM)(Linker)(CM)(Linker)(AB).

TABLE 46 PFIL2-CLIg-HK-anti-VegF 306 KK1203 LCGgccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaaggtactggccgtggtccaagctgggttggcagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 318)Linker         MM          Linker         CM   Linker     AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(GTGRGPSWVGSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 319)

TABLE 47 PFIL2-CLIg-HK-antiVegF 306 KK1204 LCggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaaggtctgagcggccgttccgataatcatggcagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 320)Linker         MM          Linker        CM   Linker      AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(GLSGRSDNHGSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 321)

TABLE 48 PFIL2-CLIg-HK-antiVegF 306 KK1214 LCggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaaccactgactggtcgtagcggtggtggaggaagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 322)Linker         MM          Linker        CM    Linker      AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(PLTGRSGGGGSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 323)

TABLE 49 PFIL2-CLIg-HK-antiVegF 306 SM1215 LCggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagaaactccatctgtaaagactatgggccgtagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 324)Linker        MM           Linker      CM     Linker      ABGQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(ETPSVKTMGRSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 325)Plasmin-Activated AA Sequences

Nucleotide and amino acid sequences of plasmin enzyme-activated antiVEGF light chain AAs are provided in the tables below. Parenthesesdelineate the demarcations between the various sequence domains:(Linker)(MM)(Linker)(CM)(Linker)(AB).

TABLE 50 PFIL2-CLIg-HK-antiVegF 306 SM1239 LCggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaaggtttcccacatatgaaaactttccagcatagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 326)Linker         MM          Linker      CM     Linker      AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(GFPHMKTFQHSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 327)Legumain-Activated AAs

The sequences for the legumain substrates AANL (SEQ ID NO: 361) and PTNL(SEQ ID NO: 362) are known in the art (Liu, et al. 2003. Cancer Research63, 2957-2964; Mathieu, et al 2002. Molecular and BiochemicalParisitology 121, 99-105). Nucleotide and amino acid sequences oflegumain enzyme-activated anti VEGF light chain AAs are provided in thetables below. Parentheses delineate the demarcations between the varioussequence domains: (Linker)(MM)(Linker)(CM)(Linker)(AB).

TABLE 51 PFIL2-CLIg-HK-antiVEGF 306 AANL (SEQ ID NO: 361) Light Chainggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagcagctaatctgggcagcggaggaagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 328)Linker         MM          Linker      CM    Linker        AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(AANLGSGGSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 329)

TABLE 52 PFIL2-CLIg-HK-antiVEGF 306 PTNL (SEQ ID NO: 362) Light Chainggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaaccgactaatctgggcagcggaggaagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 330)Linker         MM          Linker       CM   Linker        AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(PTNLGSGGSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 331)

TABLE 53 PFIL2-CLIg-HK-antiVEGF 306 PTN Light Chainggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaaccgactaatggtggcagcggaggaagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 332)Linker         MM          Linker       CM   Linker        AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(PTNGGSGGSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 333)Caspase Activated AAs

Nucleotide and amino acid sequences of caspase enzyme-activated antiVEGF light chain AAs are provided in the tables below. Parenthesesdelineate the demarcations between the various sequence domains:(Linker)(MM)(Linker)(CM)(Linker)(AB). The caspase substrate, sequenceDEVD (SEQ ID NO: 334), is known in the art.

TABLE 54 PFIL2-CLIg-HK-antiVegF 306 DEVD (SEQ ID NO: 334) LCggccagtctggccagccgtgttctgagtggcagtcgatggtgcagccgcgttgctattatgggggcggttctggtggcagcggccaaggtggccaagacgaagtcgatggcagcggaggaagtagcggcggttctgatattcaactgacccagagcccttcttccctgagtgccagcgtgggtgaccgtgttacgatcacttgctcggccagccaagatatttctaactacctgaattggtaccagcagaagccaggaaaggcaccaaaagtcctgatctacttcacaagttcactgcattccggcgtaccgtcgcgctttagcggttctggcagtggtaccgacttcaccctgactatctcgagtctgcaacctgaggattttgctacatattactgtcagcaatattcgaccgtgccgtggacgttcgggcagggcaccaaagtggagattaagcgtacggtggctgcaccatctgtcttcatcttcccgccatctgatgagcagttgaaatctggaactgcctctgttgtgtgcctgctgaataacttctatcccagagaggccaaagtacagtggaaggtggataacgccctccaatcgggtaactcccaggagagtgtcacagagcaggacagcaaggacagcacctacagcctcagcagcaccctgacgctgagcaaagcagactacgagaaacacaaagtctacgcctgcgaagtcacccatcagggcctgagctcgcccgtcacaaagagcttcaacaggggagagtgttag (SEQ ID NO: 335)Linker         MM          Linker      CM    Linker        AB(GQSGQ)(PCSEWQSMVQPRCYYG)(GGSGGSGQGGQ)(DEVDGSGGSS)(GGS)(DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKRTVAAPSVFIFPPSDEQLKSGTASVVCLLNNFYPREAKVQWKVDNALQSGNSQESVTEQDSKDSTYSLSSTLTLSKADYEKHKVYACEVTHQGLSSPVTKSFNRGEC) (SEQ ID NO: 336)Construction of Legumain and Caspase Activated AA Expression Vectors

Substrates were constructed in a two step process. First, two productswere PCR amplified using the CX0325 forward primer with a substratespecific reverse primer (CX0720 AANL (SEQ ID NO: 361), CX0722 PTNL (SEQID NO: 362), CX0724 PTN, and CX0758 DEVD (SEQ ID NO: 334)), the otherPCR amplified using the CX0564 reverse primer with a substrate specificforward primer (CX0721 AANL (SEQ ID NO: 361), CX0723 PTNL (SEQ ID NO:362), CX0725 PTN, and CX0754 DEVD (SEQ ID NO: 334). In both cases thesubstrate for the PCR was the anti-VEGF mmp-9 306 scFv. Second, the twoproducts were combined and PCR amplified using the outside primersCX0325 and CX0564. The final products were cloned into thepFIL2-CL-anti-VEGF Lc using the EcoRI and XhoI restriction sites.

TABLE 55 Primers for Construction of Legumain and Caspase Activated AAexpression Vectors CXO564 aggttgcagactcgagatagtcagggtgaagtc (SEQ ID NO:337) CX0720 tcctccgctgcccagattagctgcttggccaccttggccgctgccac (SEQ ID NO:338) CX0721 gcagctaatctgggcagcggaggaagtagcggcggttctgatattcaactg (SEQ IDNO: 339) CX0722 tcctccgctgcccagattagtcggttggccaccttggccgctgccac (SEQ IDNO: 340) CX0723 ccgactaatctgggcagcggaggaagtagcggcggttctgatattcaactg (SEQID NO: 341) CX0724 tcctccgctgccaccattagtcggttggccaccttggccgctgccac (SEQID NO: 342) CX0725 ccgactaatggtggcagcggaggaagtagcggcggttctgatattcaactg(SEQ ID NO: 343) CX0754gacgaagtcgatggcagcggaggaagtagcggcggttctgatattcaactg (SEQ ID NO: 344)CX0758 tcctccgctgccatcgacttcgtcttggccaccttggccgctgccac (SEQ ID NO: 345)Expression and Purification of Legumain Activated AAs

3 μg of pFIL-VEGF-HL and 3 μg pFIL2-306-substrate-VEGF-light wereco-transfected into CHO—S cells (Invitrogen) using Lipofectamine 200(Invitrogen) according to manufacturers protocol. Transfected cells werecultured in Freestyle CHO media (Invitrogen) and selected for resistanceto zeocin and blasticidin. Individual clones were isolated by limitingdilution and selected for expression of human IgG capable of bindingEGFR by ELISA. All antibodies and AAs are purified by Protein-Achromatography using standard techniques.

Assay Description for the scFv AA Digest

ScFv AAs were diluted to 200 nM in assay buffer and combined withrhLegumain diluted in assay buffer at 2 ug/ml. Digests were incubatedovernight at room temperature. IgG AAs were diluted to 200 nM in assaybuffer and combined with rhLegumain diluted in assay buffer atconcentrations form 2-40 mg/mL (final rhLegumain concentrations 1 μg/ml,5 μg/ml, 20 μg/ml. Digests were incubated overnight a 37° C. Followingdigestion, the extent of activation was measured by the extent of AAbinding to VEGF on ELISA plates, visualized with anti-human-Fc. FIG. 27Panel A shows activation of ScFv AAs containing legumain substrates AANL(SEQ ID NO: 361) and PTNL (SEQ ID NO: 362) following treatment with 5mg/mL legumain. Panel B shows activation of an anti-VEGF IgG AAcontaining the legumain substrate PNTL.

In Vivo Stability of Legumain Activated AAs

Four 12 week old Balb/C mice were each given a single bolus injection of100 μg of a plasmin activated AA, AA^(PLAS)VEGF, or one of the legumainactivated AAs, AA^(AANL (SEQ ID NO: 361))VEGF orAA^(PTNL (SEQID NO: 362))VEGF. At 15 minutes, 1 day, 3 days, and 7 daysfollowing injection, serum was collected. Total AA concentration wascalculated from ELISA measurement of total human Fc in the serum. Theconcentration of activated antibody was calculated from a human VEGFbinding ELISA measurement and is shown in FIG. 28. Legumain activatedAAs isolated from serum up to 7 days following injection remain masked.(n=4). The ratio of activated AA to total AA at each time point is shownin FIG. 28 as the average of measurements from individual animals and isexpressed as percent activated. While the plasmin activated AA is nearlycompletely activated at 7 days both legumain activated AAs are onlyminimally activated.

Example 18: Serum Half Lives of AAs

FIG. 29 shows that a masked single-chain Fv-Fc fusion pro-antibodiesexhibit increased serum half-life. A masking polypeptide is appended toan antibody N-terminus such that the mask can interact with the antibodycombining site to increase thermodynamic stability or block neutralizingantibodies. A protease substrate can be used to enable removal of themask at different rates in serum or specific tissues.

FIG. 30 shows that the scFv-Fc serum concentration in healthy mice over10 days. C57Bl/6 mice (n=3 per time point) were given a single dose (150μg) of anti-VEGF scFv-Fc, AA ^(MMP)VEGF (AA 1) or AA ^(Plasmin)VEGF (AA2). Serum was collected at the indicated times and the concentration oftotal scFv-Fc was measured by ELISA. The AA concentration remainedstable 7 days post does, whereas the parent scFv-Fc concentrationdecreased after 3 days and was almost undetectable at 10 days.

FIG. 31 shows that AA scFv-Fc concentrations are elevated and persistlonger in serum compared with parent scFv-Fc in tumor-bearing mice: Anequivalent single dose of anti-VEGF scFv-Fc, AA ^(MMP)VEGF (AA 1) or AA^(Plasmin)VEGF (AA 2) was given Nude mice bearing HT29 xenografts (A) orMDA-MB-231 xenografts (B). Serum was collected at the indicated timesand the concentration of total scFv-Fc was measured by ELISA. In bothstudies a higher percentage of the initial AA dose was detected in theserum at 3 days (B) and 3 and 7 days (A).

FIG. 32 shows that AAs persist at higher concentrations in a multidosestudy: Tumor-bearing Balb/c nu/nu mice were injected with 5 mg/kg ofparental VEGF scFv-Fc, AA1, 2 or 3 every 3 days. Serum was collected atthe indicated times and the concentration of AA or parent scFv-Fc wasmeasured by ELISA. All three AAs maintained significantly higher serumconcentrations than the parent throughout the study.

Amino Acid Sequences of VEGF scFv-Fcs AAs

TABLE 56 The amino acid sequence of Anti-VEGF scFv-Fc from which AAscFvs were derived DIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGSGGGSGGGSGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPGK (SEQ ID NO: 346)

TABLE 57 AA^(MMP)VEGF: AAs contain a masking peptide and MMP substrateattached by a short linker as shown maskingpeptide                   substrateGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQVHMPLGFLGPGGSDIQLTQSPSSLSA SVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGSGGGSGGGSGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSP GK (SEQ IDNO: 347)

TABLE 58 AA^(PLASMIN)VEGF: AAs contain a masking peptide and Plasminsubstrate attached by a short linker as shown maskingpeptide                   substrateGQSGQPCSEWQSMVQPRCYYGGGSGGSGQGGQQGPMFKSLWDGGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQKSLSLSPG K (SEQ ID NO:348)

TABLE 59 AA^(NoSubstrate)VEGF AAs contain a masking peptide, Gly Ser(GS) linkers and VEGF but no substrate attached by a short linker asshown masking peptide                   substratePCSEWQSMVQPRCYYGGSGGGSGQSGQGGSGGSGQGGQGSDIQLTQSPSSLSASVGDRVTITCSASQDISNYLNWYQQKPGKAPKVLIYFTSSLHSGVPSRFSGSGSGTDFTLTISSLQPEDFATYYCQQYSTVPWTFGQGTKVEIKGGGSGGGSGGGSGGGGSGGGGSGGGGSGEVQLVESGGGLVQPGGSLRLSCAASGYDFTHYGMNWVRQAPGKGLEWVGWINTYTGEPTYAADFKRRFTFSLDTSKSTAYLQMNSLRAEDTAVYYCAKYPYYYGTSHWYFDVWGQGTLVTVSGGSGAMVRSDKTHTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEGLHNHYTQK SLSLSPGK (SEQID NO: 349)AAs Exhibit Increased Serum Half-Life as Compared to the ParentalAntibody

Eight 12 week old Balb/C mice were each given a single bolus injectionof 100 μg of a MMP activated AA, AA^(MMP)VEGF, a Plasmin activated AA,AA^(PLAS)VEGF or parental anti-VEGF antibody Ab-VEGF. At 15 minutes, 8hours, 1 day, 3 days, 7 days and 10 days following injection, serum wascollected. Total AA concentration was calculated from ELISA measurementof total human Fc in the serum. The concentration of total AA at eachtime point is shown in FIG. 33 as the average of measurements fromindividual animals and is expressed as percent of initial dose. The AAsand parental antibody distribute similarly and as expected. Reaching ahigh and equal concentration at 15 minutes and distributing into thetissues over the first day. In contrast to the parental antibody whichis nearly completely eliminated over 10 days, both AAs persist at higherlevels in the serum for the duration of the experiment.

Example 19: Reduction in Side Effects Upon Administration of an AA

Greater than 80% of the patients typically administered a conventionalEGFR antibody therapeutic exhibit toxicity of the skin, the largestorgan of the body. When patients are administered AAs directed againstEGFR it is expected that there will be little or no toxicity of theskin, as the AA will not be activated in the skin, due to lack ofdisease specific CM. As such, it is expected that the anti-EGFR AB ofthe AA will not be able to specifically bind the EGFR target.Additionally it is expected that in such patients, because the AA willnot be active in the skin, the AA will not be sequestered and it isexpected that the serum levels of the AA will remain high, therebyincreasing the concentration of the AA in the diseased tissue,effectively raising the effective dose. Hydrolysis of the CM in thediseased tissue based on the disease environment will lead to anactivated AA allowing for unmasking and specific binding of the AB tothe EGFR target, and will lead to the desired therapeutic effect.

While the present invention has been described with reference to thespecific embodiments thereof, it should be understood by those skilledin the art that various changes may be made and equivalents may besubstituted without departing from the true spirit and scope of theinvention. In addition, many modifications may be made to adapt aparticular situation, material, composition of matter, process, processstep or steps, to the objective, spirit and scope of the presentinvention. All such modifications are intended to be within the scope ofthe claims appended hereto.

What is claimed is:
 1. A method of treating or delaying the progressionof an EGFR-related disorder in a subject comprising: administering atherapeutically effective amount of an activatable antibody to a subjectin need thereof, wherein the activatable antibody, which in an activatedstate binds Epidermal Growth Factor Receptor (EGFR), comprises: anantibody or an antigen binding fragment thereof (AB) that specificallybinds to EGFR, wherein the AB comprises the heavy chain complementaritydetermining regions (CDRs) and the light chain CDRs of cetuximab; amasking moiety (MINI) coupled to the AB that inhibits the binding of theAB of the activatable antibody in an uncleaved state to EGFR, whereinthe MM comprises an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, 218-220, and 236-266; and at least onecleavable moiety (CM) coupled to the AB, wherein the CM is a polypeptidethat functions as a substrate for a protease.
 2. The method of claim 1,wherein the MM comprises the amino acid sequence CISPRGC (SEQ ID NO: 1).3. The method of claim 1, wherein the MM comprises the amino acidsequence CISPRGCPDGPYVM (SEQ ID NO: 218) or CISPRGCPDGPYVMY (SEQ ID NO:238).
 4. The method of claim 1, wherein the MM does not interfere orcompete with the AB for binding to EGFR when the activatable antibody isin a cleaved state.
 5. The method of claim 1, wherein the MM is apolypeptide of no more than 40 amino acids in length.
 6. The method ofclaim 1, wherein in the presence of EGFR, the MM reduces the ability ofthe AB to bind EGFR by at least 90% when the CM is uncleaved, ascompared to when the CM is cleaved when assayed in vitro using a targetdisplacement assay.
 7. The method of claim 1, wherein the protease isco-localized with EGFR in a tissue, and wherein the protease cleaves theCM in the activatable antibody when the activatable antibody is exposedto the protease.
 8. The method of claim 1, wherein the CM is apolypeptide of up to 15 amino acids in length.
 9. The method of claim 1,wherein the CM is a substrate for an enzyme selected from the groupconsisting of the enzymes in Table
 3. 10. The method of claim 1, whereinthe CM is a substrate for an enzyme selected from the group consistingof a urokinase-type plasminogen activator (uPA), a legumain, amatriptase, and an MMP.
 11. The method of claim 1, wherein the CMcomprises an amino acid sequence selected from the group consisting ofTGRGPSWV (SEQ ID NO: 267); SARGPSRW (SEQ ID NO: 268); TARGPSFK (SEQ IDNO: 269); TARGPSW (SEQ ID NO: 270), LSGRSDNH (SEQ ID NO: 271); GGWHTGRN(SEQ ID NO: 272); HTGRSGAL (SEQ ID NO: 273); PLTGRSGG (SEQ ID NO: 274);LTGRSGA (SEQ ID NO: 275); AARGPAIH (SEQ ID NO: 276); RGPAFNPM (SEQ IDNO: 277); SSRGPAYL (SEQ ID NO: 278); RGPATPIM (SEQ ID NO: 279); RGPA(SEQ ID NO: 280), GLSGRSDNHGSS (SEQ ID NO: 366); PLTGRSGGGGSS (SEQ IDNO: 367); ETPSVKTMGRSS (SEQ ID NO: 368); and GTGRGPSWVGSS (SEQ ID NO:369).
 12. The method of claim 1, wherein the CM comprises the amino acidsequence LSGRSDNH (SEQ ID NO: 271).
 13. The method of claim 1, whereinthe activatable antibody in the uncleaved state has the structuralarrangement from N-terminus to C-terminus as follows: MM-CM-AB orAB-CM-MM.
 14. The method of claim 1, wherein the activatable antibodycomprises a linking peptide between the MM and the CM.
 15. The method ofclaim 1, wherein the activatable antibody comprises a linking peptidebetween the CM and the AB.
 16. The method of claim 1, wherein theactivatable antibody comprises a first linking peptide (LP1) and asecond linking peptide (LP2), and wherein the activatable antibody inthe uncleaved state has the structural arrangement from N-terminus toC-terminus as follows: MM-LP1-CM-LP2-AB or AB-LP2-CM-LP1-MM.
 17. Themethod of claim 16, wherein the two linking peptides need not beidentical to each other.
 18. The method of claim 16, wherein each of LP1and LP2 is a peptide of about 1 to 20 amino acids in length.
 19. Themethod of claim 16, wherein at least one of LP1 or LP2 comprises anamino acid sequence selected from the group consisting of (GS)n, (GGS)n,(GSGGS)n (SEQ ID NO: 12) and (GGGS)n (SEQ ID NO: 13), where n is aninteger of at least one.
 20. The method of claim 16, wherein at leastone of LP1 or LP2 comprises an amino acid sequence selected from thegroup consisting of GGSG (SEQ ID NO: 14), GGSGG (SEQ ID NO: 15), GSGSG(SEQ ID NO: 16), GSGGG (SEQ ID NO: 17), GGGSG (SEQ ID NO: 18), and GSSSG(SEQ ID NO: 19).
 21. The method of claim 16, wherein the activatableantibody is selected from the group consisting of: an activatableantibody wherein LP1 comprises the amino acid sequence GGSGGS (SEQ IDNO: 111), an activatable antibody wherein LP2 comprises the amino acidsequence GS or GSSG, and an activatable antibody wherein LP1 comprisesthe amino acid sequence GGSGGS (SEQ ID NO: 111) and LP2 comprises theamino acid sequence GS or GSSG.
 22. The method of claim 16, wherein theCM is a substrate for an enzyme selected from the group consisting of aurokinase-type plasminogen activator (uPA), a legumain and a matriptase,wherein the MM comprises the amino acid sequence CISPRGC (SEQ ID NO: 1),wherein LP1 comprises the amino acid sequence GGSGGS (SEQ ID NO: 111),and wherein LP2 comprises the amino acid sequence GS or GSSG.
 23. Themethod of claim 1, wherein the AB has a dissociation constant of at most100 nM for binding to EGFR.
 24. The method of claim 1, wherein theantigen binding fragment thereof is selected from the group consistingof a Fab fragment, a F(ab′)2, fragment, a scFv, a scAb, a dAb, a singledomain heavy chain antibody, and a single domain light chain antibody.25. The method of claim 1, wherein the AB is cetuximab or wherein the ABis an antigen binding fragment of cetuximab.
 26. The method of claim 1,wherein the AB is conjugated to an agent.
 27. The method of claim 26,wherein the agent is a therapeutic agent.
 28. The method of claim 27,wherein the agent is an antineoplastic agent.
 29. The method of claim26, wherein the agent is a toxin or a fragment thereof.
 30. The methodof claim 26, wherein the agent is an agent selected from the groupconsisting of the agents in Table
 4. 31. The method of claim 26, whereinthe agent is conjugated to the AB via a linker.
 32. The method of claim31, wherein the linker is a cleavable linker.
 33. The method of claim31, wherein the linker is a non-cleavable linker.
 34. The method ofclaim 1, wherein the activatable antibody comprises a detectable moiety.35. The method of claim 34, wherein the detectable moiety is adiagnostic agent.
 36. The method of claim 1, wherein the serum half-lifeof the activatable antibody is at least 5 days when administered to anorganism.
 37. The method of claim 1, wherein the CM is cleaved by aprotease in a target tissue.
 38. The method of claim 1, wherein the CMis cleaved by a protease that is co-localized with EGFR in the targettissue.
 39. The method of claim 1, wherein the EGFR-related disorder isa cancer.
 40. The method of claim 26, wherein the EGFR-related disorderis a cancer.